Merginol 901

Endpoint summary

Description of key information

No repeated dose toxicity data on the substance itself is available. Therefore, the sub-chronic toxicity study on the structural analogue with CAS# 151661-88-0 was used. Based on these observations the NOAEL was determined to be 1000 mg/kg bw/day in male and female rat. However, since a developmental toxicity study is available for a close analogue, in which the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/day, this value is adapted as starting point for hazard assessment, as a conservative approach.

To further improve the toxicological data available for this substance and to further improve the read-across hypothesis, an OECD 422 for substance itself is ordered. This study can then be used as bridging study and further support the read-across hypothesis. Based on the results obtained in a 2 week preliminary oral toxicity study in rats, it can be concluded that the oral treatment with the test substance at dosages of 300 and 1000 mg/kg/day was well tolerated although changes were noted in the absolute and relative liver weights of the highest dose.

Additionally, a subchronic study is ordered for a read-across substance (CAS 85586-35-2) to improve the available data on subchronic toxicity based on the agreed testing strategy.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990-06-18 until 1990-09-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Application was performed on 5 days per week as opposed to 7 days per week with no analytical verification of test concentrations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material: Stabiol VP 1711
- Substance type: fatty acid ester
- Physical state: white powder
- Lot/batch No.: 041/8/055 (1988-02-04)
- Stability under test conditions: stable under test conditions

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 4 weeks
- Weight at study initiation: 62 - 82 g (females); 58 - 80 g (males)
- Housing: Makrolon Cage type III
- Diet: Altromin 1324, ad libitum, supplied by Altromin GmbH
- Water: tap water (drinking water quality analytically verified), ad libitum
- Acclimation period: 13 days

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The testing solution was prepared daily just before administration by weighing out an appropriate amount of test substance and dispersing it in the vehicle.

VEHICLE
- The administration volume was 5 mL/kg bw.
- The concentration of test item in the vehicle was: 200, 60, 20 and 0 mg/mL

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

107 to 108 days starting with animal acclimation, with a total treatment of 68 to 69 administrations

Frequency of treatment:

5 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

10 animals/ sex/ dose.
Satellite groups of 5 male and 5 female animals were used for the control and 1000 mg/kg bw day dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum.
- The total number of application was 68 to 69, corresponding to a total administered amount of 6800 to 6900, 20400 to 20700, 68000 to 69000 mg/kg bw for the respective 100, 300, 1000 mg/kg bw/day dose groups. -
- Satellite groups of 5 male and 5 female animals were used for the control and 1000 mg/kg bw day dose groups. Following concurrent treatment these satellite animals were not treated for an additional observation period of 33 days in order to investigate the reversibility of potential toxic responses.

Positive control:

Not required

Observations and examinations performed and frequency:

MORTALITY
A check for moribund and dead animals was made twice daily on working days. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
All animals were checked twice daily for any abnormal clinically signs on working days.

FOOD CONSUMPTION
Group food consumption was determined weekly for each cage.

DRINKING WATER CONSUMPTION
Drinking water consumption was determined weekly for each cage.

BODY WEIGHT
Body weight was determined before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals.

HAEMATOLOGY
The following parameters were determined in blood: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Platelet count (PLT), Differential blood count

CLINICAL CHEMISTRY
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (AP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Cholesterol (CHOL)

OPHTHALMOSCOPY
Prior to the start of necropsy the eyes of all animals of the control and high dose animals were treated with Mydriaticum and examined for any changes using a slit lamp.

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by exsanguination under an overdose of ether. The exsanguinated
animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings on following organs: Adrenal glands, Aorta, Skeletal muscle, Brain, Cecum, Uterus, Coagulating glands, Colon, Duodenum, Epididymis (left), Esophagus, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Pancreas, Tounge, Pituitary gland, Prostate, Salivary gland, Seminal vesicles, Skin, Semen vesicles, Spleen, Forestomach, Testis, Thymus, Thyroid glands, Trachea, Uterus

Statistics:

- A comparison of inter-group differences (clinical chemistry, hematology and body weight) was performed using the t-test.
- DUNNETT's test was used to test the hypothesis of equal means.
- Steel-test was used to evaluate the inter-group differences relating to organ weights of each dose group.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed

Mortality:

no mortality observed

Description (incidence):

In the present study no animal died ahead of schedule.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed in all parameters under investigation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed in all parameters under investigation.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decrease of the haematocrit (HCT) value in the male groups 2 - 4 was observed. The decrease of the HCT seems to be compound-and partially dose-related. Additionally the intermediate analysis showed slightly reduced Red Blood Cell (RBC)-value for the male group 4. The observed deviation of the HCT is considered to be incidental/ because the diagnosed values are within the ranges of the historical control. In addition to this, there are no corresponding deviations of the RBC- or MCV-values to prove the biological relevance of this findings.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed in all parameters under investigation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- Absolute organ weights: No test substance-related findings were observed in all parameters under investigation.
- Relative organ weights: No test substance-related findings were observed in all parameters under investigation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopical examination of the organs displayed some observations like discolouration of the thymus deformation of the spleen, cyst of the kidney, hydronephrosis, atrophy of the testes, hydrometra and necrosis of the fatty tissue which were considered to be spontaneous. Compound-related macroscopical effects were not observed. The male and female animals of the recovery group 4 (recovery period 33 days) showed no macroscopical compound-related alterations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related findings were observed in all parameters under investigation. However, in the male and female animals of all groups (including the recovery group 1 and 4) the livers, the heart and the mandibulary lymph node showed effects which were due to a bacteriosis of unknown etiology. The observed germinal hyperplasia of the mandibulary lymph node can be interpreted as a consequence of this bacteriosis.

Histopathological findings: neoplastic:

no effects observed

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: No adverse effects observed at the highest dose tested.

Critical effects observed:

no

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985-09-23 until 1985-11-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Application was performed on 5 days per week as opposed to 7 days per week

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material: LA 990
- Substance type: organic
- Physical state: viscous liquid, beige-coloured
- Lot/batch No.: 37-4-222
- Stability under test conditions: stable under test conditions
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 4 weeks
- Weight at study initiation: 60- 81 g (females); 59 - 78 g (males)
- Housing: Makrolon Cage type III
- Diet: Altromin 1324 - ad libitum- supplied by Altromin GmbH
- Water: tap water (drinking water quality analytically verified) - ad libitum
- Acclimation period: 10 days

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxymethylcellulose and 0.5% Cremophor (CMCC)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The testing suspension was prepared by weighing out an appropriate amount of test substance and dispersing it in the vehicle.

VEHICLE
- The test item was applied as a suspension in CMCC (vehicle).
- The administration volume was 10 mL/kg body weight.
- The concentration of test item in the vehicle was: 100, 50, 10 and 0 mg/mL.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

42 to 43 days starting with animal acclimation, with a total treatment of 23 to 24 administrations

Frequency of treatment:

5 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

10 animals
Satellite groups of 5 male and 5 female animals were used for the control and 1000 mg/kg bw day dose groups. Following concurrent treatment these satellite animals were not treated for an additional observation period of 28 days in order to investigate the reversibility of potential toxic responses

Control animals:

yes, concurrent vehicle

Details on study design:

- On the day of arrival the animals were subjected to an acclimation period during which they received ground diet and drinking water ad libitum.
- The total number of application was 23 to 24, corresponding to a total administerd amount of 2300 to 2400, 11500 to 12000, 23000 to 24000 mg/kg bw for the respective 100, 500, 1000 mg/kg bw/day dose groups.
- Satellite groups of 5 male and 5 female animals were used for the control and 1000 mg/kg bw day dose groups. Following concurrent treatment these satellite animals were not treated for an additional observation period of 28 days in order to investigate the reversibility of potential toxic responses

Positive control:

Not required

Observations and examinations performed and frequency:

MORTALITY
A check for moribund and dead animals was made twice daily on working days. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
All animals were checked twice daily for any abnormal clinically signs on working days.

FOOD CONSUMPTION
Group food consumption was determined weekly for each cage.

DRINKING WATER CONSUMPTION
Drinking water consumption was determined weekly for each cage.

BODY WEIGHT
Body weight was determined before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals.

HAEMATOLOGY
The following parameters were determined in blood: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Platelet count (PLT), Differential blood count,

CLINICAL CHEMISTRY
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (AP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Cholesterol (CHOL)

OPHTHALMOSCOPY
Prior to the start of necropsy the eyes of all animals of the control and high dose animals were treatd with Mydriaticum and examined for any changes using a slit lamp.

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by exsanguination under an overdose ether. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings on following organs: Adrenal glands, Aorta, Skeletal muscle, Brain, Cecum, Uterus, Coagulating glands, Colon, Duodenum, Epididymis (left), Esophagus, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Pancreas, Tounge, Pituitary gland, Prostate, Salivary gland, Seminal vesicles, Skin, Semen vesicles, Spleen, Forestomach, Testis, Thymus, Thyroid glands, Trachea, Uterus

Statistics:

- A comparison of inter-group differences (clinical chemistry, hematology and body weight) was performed using the t-test.
- Steel-test was used to evaluate the inter-group differences relating to organ and bone weights of each dose group.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed

Mortality:

no mortality observed

Description (incidence):

In the present study no animal died ahead of schedule.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed in all parameters under investigation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed following substance administration - No further test substance-related findings were observed.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A decrease of the haematocrit (HCT) value and Red Blood Cell (RBC) were observed for male animals in the high dose group. In female animals the same findings occurred in the middle dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A slight significant increase in GPT-levels in males of the high dose and a significant increase of GPT-values in female animals of the high dose was measured.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative liver weights were significantly increased in male animals of the high dose. A slight increase of the relative liver weights was seen in female animals of the high dose. These findings were considered to be compound related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopical examination of the organs did not reveal any compound related changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination in animals of high dose level revealed degenerative alterations in kidneys which were considered to be reversible in the recovery group.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: see any other information on results incl tables

Critical effects observed:

no

It is argueable whether the effects observed in the study are sufficient to conclude on a NOAEL of 100 mg/kg bw/d. According to a recent publication by Hall et al., the variation of liver weights without histological evidence or distinct alterations in clinical chemistry is not considered to be an adverse effect. Hall et al. reach the conclusion that only a 2-3-fold increase in ALT (GPT) or a biologically significant change in other biomarkers is considered to be an adverse effect. In the study at hand, the only remarkable alteration in liver parameters was an ALT increase in the highest dose group of males and females, but it was less than 2-fold. Regarding the observed degradations in the kidney, they were not accompanied with relevant biochemical changes (urea, creatinie, sodium, potassium) and were reversible in the recovery group. The absence of adverse effects in the 90-day study further supports the argumentation that the alterations found in the 28-day study were temporary and rather a non-adverse adaptation than an evidence for a toxicological action.

Reference

Hall et al. (2012), Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes – conclusions from the third international ESTP workshop, Toxicol Pathol, 40(7), 971-94

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 2021 - May 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

dose finding test study

Qualifier:

no guideline available

Principles of method if other than guideline:

14-day dose-range-finding study with daily intake concentrations of 0, 300, 1000 mg/kg bw of the test substance. The test substance was administered orally by gavage at a dose volume of 10 mL/kg bw. Control animals were dosed with the vehicle alone (0.5% CarboxyMethyl Cellulose).

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 0019913739
- BASF compound no.: 12/0028-3
- Retest date: 01 Nov 2021
- The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Analysis was performed to verify the stability and the concentration of the preparations.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- the test item was dissolved in the vehicle to reach required concentrations
- preparations were made daily
- concentrations were calculated and expressed in terms of test item as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- in the vehicle: 0.5% CarboxyMethyl Cellulose (CMC)

OTHER SPECIFICS
- Appearance: Liquid, viscous yellowish, clear

Species:

rat

Strain:

other: Wistar Hannover

Details on species / strain selection:

The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 200-225 g for males and 175-200 g for females
- Fasting period before study: no data
- Housing: 4 animals of one sex per cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): water bottles, ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C±2 °C
- Humidity (%): 55%±15 %
- Air changes (per hr): 15-20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 12 April 2021 To: 04 May 2021

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Storage temperature of food: room temperature

VEHICLE
- The vehicle will be 0.5% CMC.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in order to validate the analytical method, the preparation procedure
and to verify the stability and the concentration of the preparations. Samples of the
preparations prepared on Day 1 were analysed to check the stability and concentration.
Results were within the limit of the acceptance.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor (dose range finding study)
- The rats were allocated to the 3 groups by computerised stratified
randomisation to give approximately equal initial group mean body weights.

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and public holidays, a similar procedure were followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation, twice weekly thereafter and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food consumed by each cage of rats was recorded at the same intervals as per body weight following allocation, where possible. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of sacrificial procedure
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all viable animals
- Following parameters were examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, large unstained cells), Platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of sacrificial procedure
- Animals fasted: Yes
- How many animals: all viable animals
- Following parameters were examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Urea, Creatinine, Glucose, total bilirubin, total cholesterol, total protein, Sodium, Potassium, Calcium, Chloride, Bile Acids

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Adrenal glands, Liver, Kidneys, Spleen)

HISTOPATHOLOGY: No

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be in homogeneous a Modified t test (Cochran and Cox) was applied.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was the treatment-related clinical sign observed in all treated animals of the high dose group and in two males and one female of the low dose group (see tables 1 and 2).

Mortality:

mortality observed, treatment-related

Description (incidence):

One female from the high dose group was found dead on Day 9 of the study. Dyspnoea, ataxia and decreased activity were described before death.
At macroscopic observations ruptured oesophagus was observed. The finding observed was due to a misgavage and considered the cause of death.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in the absolute and relative mean liver weights was observed in males and females at 1000 mg/kg/day. This change could be considered related to treatment (see tables 3 and 4).

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Table 1: 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS: Salivation (Males)

Males	Control		300 mg/kg bw/day		1000 mg/kg bw/day
	a	b	a	b	a	b
Salivation	0	0	2	4.5	4	8.8

a = Number of animals affected
b = Number of days with clinical sign/animal

Table 2: 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS: Salivation (Females)

Females	Control		300 mg/kg bw/day		1000 mg/kg bw/day
	a	b	a	b	a	b
Salivation	0	0	1	2	4	5.8

a = Number of animals affected
b = Number of days with clinical sign/animal

Table 3: 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS: Organ weights (Males)

Males		TBW ( g )	Adrenal glands ( g )	Kidneys ( g )	Liver ( g )	Spleen ( g )
Control	(n)	4	4	4	4	4
	Mean	315.98	0.07	1.975	7.961	0.7
	SD	14.615	0.01122	0.0751	0.3221	0.11932
300 mg/kg bw/day	(n)	4	4	4	4	4
	Mean	309.15	0.0605	1.879	7.663	0.6765
	SD	16.041	0.00777	0.2221	0.4467	0.11147
1000 mg/kg bw/day	(n)	4	4	4	4	4
	Mean	316.6	0.0743	2.195	9.506	0.7578
	SD	23.534	0.00727	0.2284	1.4122	0.10762

Table 4: 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS: Organ weights (Females)

Females		TBW ( g )	Adrenal glands ( g )	Kidneys ( g )	Liver ( g )	Spleen ( g )
Control	(n)	4	4	4	4	4
	Mean	217.73	0.0745	1.381	5.674	0.5443
	SD	9.533	0.011	0.0952	0.3771	0.02658
300 mg/kg bw/day	(n)	4	4	4	4	4
	Mean	222.88	0.0743	1.329	6.032	0.521
	SD	9.051	0.00695	0.0847	0.3133	0.07181
1000 mg/kg bw/day	(n)	3	3	3	3	3
	Mean	218.5	0.064	1.362	6.987	0.5927
	SD	10.802	0.0052	0.104	0.9534	0.09448

Conclusions:

Based on the results obtained in this study, it can be concluded that the oral treatment with the test substance at dosages of 300 and 1000 mg/kg/day for 2 weeks was well tolerated although changes were noted in the absolute and relative liver weights of the highest dose.

Executive summary:

The toxicity of the test substance was investigated in Wistar Han rats after daily oral administration for 2 weeks. Two groups of 4 male and 4 female rats received the test item at dose levels of 300 and 1000 mg/kg/day. A third, similarly consituted group received the vehicle alone (0.5% CMC) and acted as a control. One female rat receiving the high dose level was found dead following a misdosing. No relevant changes were recorded in clinical pathology parameters after 2 weeks of treatment. Animals of both gender showed an increase in the absolute and relative mean liver weights at 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data on repeated dose toxicity of the substance itself are available. Therefore, investigations on repeated dose toxicity of the structural analogous substances with CAS# 151661-88-0 and Oleic methyl ester, epoxidized, reaction products with glycerol (OEG) were used. Since a developmental toxicity study is available the close analogue with CAS# 211450 -54 -3, in which the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/day, this value is adapted as starting point for hazard assessment, as a conservative approach.

To further improve the toxicological data available for this substance and to further improve the read-across hypothesis, an OECD 422 for substance itself is ordered. This study can then be used as bridging study and further support the read-across hypothesis.

 

2 week preliminary oral toxicity study in rats

The toxicity of the test substance was investigated in Wistar Han rats after daily oral administration for 2 weeks. Two groups of 4 male and 4 female rats received the test item at dose levels of 300 and 1000 mg/kg/day. A third, similarly consituted group received the vehicle alone (0.5% CMC) and acted as a control. One female rat receiving the high dose level was found dead following a misdosing. No relevant changes were recorded in clinical pathology parameters after 2 weeks of treatment. Animals of both gender showed an increase in the absolute and relative mean liver weights at 1000 mg/kg/day. Based on the results obtained in this study, it can be concluded that the oral treatment with the test substance at dosages of 300 and 1000 mg/kg/day was well tolerated.

 

90-day repeated dose toxicity (CAS 151661-88-0)

The substance was tested for systemic toxicity at repeated doses of 0 (group 1), 100 (group 2), 300 (group 3) and 1000 (group 4) mg/kg body weight/ day. The compound was administered daily by gavage over a period of 90 days. 10 male and 10 female rats were used for each dose. In addition to the groups 1 and 4, 5 male and 5 female animals were used to determine the reversibility of passible compound- related findings (recovery groups). All doses applied were tolerated without lethality. No compound- related symptoms were observed. The mean food consumption in all treated groups was comparable to the control. The increase in food consumption in the female group 4 at the beginning of the week 4 up to the end resulted from food wasting by the animals. The mean water intake of all groups was comparable to the control. The total body weight gain of all groups showed no deviation and was comparable to the control. The intermediate and the final haematological examinations revealed a decrease of the Haematocrit value (HCT) in the male groups 2 - 4. The decrease of the HCT seems to be compound- and partially dose-related. Additionally the intermediate analysis showed slightly reduced (Red Blood Cell count) RBC- value for the male group 4. The observed deviation of the is considered to be incidental/ because the diagnosed values are within the limits of the historical control. In addition to this, there are no corresponding deviations of the RBC- or MCV- values to prove the biological relevance of this findings. All other findings were considered to be spontaneous and therefore not related to the treatment. The biochemical examinations revealed some compound and dose independent findings. The examination of the eyes by slit lamp microscope showed no compound- related effects. The absolute and relative organ weights in all groups showed no deviations and were comparable to the control. The macroscopical examination of the organs displayed some observations like discolouration of the thymus, deformation of the spleen, cyst of the kidney, hydronephrosis, atrophy of the testes, hydrometra and necrosis of the fatty tissue which were considered to be spontaneous. Compound-related macroscopical effects were not observed. The male and female animals of the recovery group 4 (recovery period 33 days) showed no macroscopical compound- related alterations. The microscopical examination revealed no compound- related effects. In the male and female animals of all groups (including the recovery group 1 and 4 ) the livers, the heart and the mandibulary lymph node showed effects which were due to a bacteriosis of unknown etiology. The observed germinal hyperplasia of the mandibulary lymph node can be interpreted as a consequence of this bacteriosis. Possible target organs have not been diagnosed. The liver and the heart of the recovery groups 1 and 4 were examined in order to prove the persistence of the bacteriosis. According to the described study, a daily administration of the test item up to 1000 mg/kg bw/day is not cumulative- systemic toxic to rats. Based on these observations the NOAEL was determined to be 1000 mg/kg bw/day for males and females(Henkel 1991).

 

28-day repeated dose toxicity (Oleic methyl ester, epoxidized, reaction products with glycerol (OEG))

The substance was tested for systemic toxicity at dosages of 100, 500 and 1000 mg/kg body weight/day, administered by gavage for 28 days. 10 male and 10 female rats were used for each dose. The low, middle and high dose was tolerated in all animals without administration related lethality. The only symptom was salivation in few animals of the high dose group. There were some variations in food, water consumption and body weight gain in dosage groups to be without compound and dose related relevance. Haematologically, compound related, decreased haematocrit and erythrocyte values were observed in male animals in the high dose group. In female animals the same findings occurred in the middle dose group. A slight significant increase in GPT-levels in males of the high dose and a significant increase of GPT-values in female animals of the high dose were measured. Eye examination revealed no compound related findings. The relative liver weights were significantly increased in male animals of the high dose. A slight increase of the relative liver weights was seen in female animals of the high dose. These findings were considered to be compound related. Pathologically-anatomically examination revealed in all animals no evidence of compound related damage of inner organs. Microscopic examination in animals of high dose level revealed degenerative alterations in kidneys which were reversible in the recovery group. It was concluded by the study director that a daily administration of 1000 mg/ kg bw/ day is for rats a cumulative toxic level with liver and kidney as target organs. A NOAEL of 100 mg/kg bw/day was suggested as non-cumulative toxic level for rats (BASF 1987).

However, it is arguable whether the effects observed in the study are sufficient to conclude on a NOAEL of 100 mg/kg bw/day. According to a recent publication by Hall et al., the variation of liver weights without histological evidence or distinct alterations in clinical chemistry is not considered to be an adverse effect. Hall et al. reach the conclusion that only a 2-3-fold increase in ALT (GPT) or a biologically significant change in other biomarkers is considered to be an adverse effect. In the study at hand, the only remarkable alteration in liver parameters was an ALT increase in the highest dose group of males and females, but it was less than 2-fold. Regarding the observed degradations in the kidney, they were not accompanied with relevant biochemical changes (urea, creatinine, sodium, potassium) and were reversible in the recovery group. The absence of adverse effects in the 90-day study further supports the argumentation that the alterations found in the 28-day study were temporary and rather a non-adverse adaptation than an evidence for a toxicological action (Henkel 1987).

 

Reference

Hall et al. (2012), Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes – conclusions from the third international ESTP workshop, Toxicol Pathol, 40(7), 971-94

 

Justification for classification or non-classification

Based on the available data, classification for repeated dose toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.