Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

Endpoint summary

Description of key information

Based on existing datasets and structural and chemical considerations, read-across from the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate to available repeated dose toxicity studies on isobutanol and isobutyl isobutyrate is appropriate to meet the REACH Annex VII-IX data requirements. In a sub-chronic, 90 day toxicity study using isobutanol in which rats were treated by oral gavage at 0,100,316 or 1000 mg/kg bw/day, the NOAEL was determined to be 316 mg/kg bw/day based on decreased bodyweight gain and transient hypoactivity and ataxia at the higher dose. The sub-chronic oral toxicity of isobutyl isobutyrate was determined to be low: the NOAEL was 1000 mg/kg bw/day (i.e. the highest dose tested). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July - 6 November 1985

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Groups of male and female rats were dosed orally once daily with 0, 100, 316, or 1000 mg/kg/day of the compound until the day of necropsy at an interim or final sacrifice (days 29 and 30 or days 92 and 93 respectively). The first 10 male and 10 female rats from each group were scheduled for the interim necropsy and all remaining rats were sacrificed on day 92 or 93.
Body weights, food consumption, mortality, any overt signs of toxicity and ophthalmologic examinations were carried out on all animals. Blood and urine were collected from a fifth group of 10 rats/sex prior to initiation of dosing, from all surviving rats at the interim sacrifice and from 10 rats/sex/group at the final sacrifice for clinicopathologic evaluation.
Gross postmortem examinations were done on all rats and organ weights were obtained at the interim and final sacrifices. A histopathologic examination was done on all rats in the control and high-dose groups, on rats found dead or sacrificed moribund, on the livers, hearts, and kidneys of low- and mid-dose rats, and on all gross lesions.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 22-23 days
- Weight at study initiation: 45-55 grams
- Fasting period before study: no
- Housing: individually in wire-botton cages
- Diet (e.g. ad libitum): Purina Certified Rodent Laboratory Chow No. 5002 (pellet) ad libitum
- Water (e.g. ad libitum): Filtered municipal water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° F ±2 (22 ±1 °C )
- Humidity (%): 43% ± 5
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): A 12 hour light: 12 hour dark cycle

IN-LIFE DATES: From: Not reported To: Not reported

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh solutions of the compound (in deionized water) were prepared weekly and dosed orally at a volume of 10 ml/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Laboratory analyses done by the County of Muskegon, Wastewater Management System

Duration of treatment / exposure:

29 and 30 days for rats subject to interim necropsy
92 and 93 for the remaining rats subject to final necropsy

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100, 316, or 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Four groups 30 rats/sex/group and a fifth group of 10/sex

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

The rats were assigned randomly to groups.
No further data on dose selection rationale reported.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily 7 days a week
- Cage side observations: mortality and clinical effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily 7 days a week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the pretreatment period and week 13
- Dose groups that were examined: All rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to initiation of dosing from group V, during week 4 or 5 from interim sacrifice rats, during week 13 or 14 from final sacrifice rats
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: No
- How many animals: 10 male and 10 female rats in group V, all surviving rats scheduled for the interim, and the first 10 (numerically) surviving males and first ten surviving females from each group at the final sacrifice
- Parameters checked: haemoglobin, hematocrit, erythrocyte count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total and differential leucocyte counts, estimated platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to initiation of dosing from group V, during week 4 or 5 from interim sacrifice rats, during week 13 or 14 from final sacrifice rats
- Animals fasted: No
- How many animals: 10 male and 10 female rats in group V, all surviving rats scheduled for the interim, and the first 10 (numerically) surviving males and first ten surviving females from each group at the final sacrifice
- Parameters checked: alkaline phosphatase, blood urea nitrogen, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, glucose,
total protein, albumin, A/G ratio (calculated), globulin (calculated), total bilirubin, sodium, potassium, chloride, calcium, creatinine, inorganic phosphate, total carbon dioxide, total serum cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to initiation of dosing from group V, during week 4 or 5 from interim sacrifice rats, during week 13 or 14 from final sacrifice rats
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: pH, specific gravity, glucose, protein, bilirubin, ketones, urobilinogen, microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
A necropsy was performed on the surviving rats of the first 10 males and 10 females from each dose group on day 29 or 30 of the study. On days 92 or 93 the remaining rats were sacrificed. Rats found dead were necropsied immediately after they were found.
At the interim and final sacrifices, rats were anesthetized with CO2 and exsanguinated by cardiac puncture. The cranial, thoracic and peritoneal cavities were opened and the contents examined macroscopically. The organs and tissues listed in table 1 were removed from each animal and preserved.
Lungs of rats at the scheduled sacrifice were inflated with formalin via the trachea. Eyes with attached optic nerve from all rats killed at the interim and final sacrifices were preserved in a modified Zenker's fixative. The testes with attached epididymides of all male rats were preserved in Bouin's fixative. All other tlssues were preserved in 10% neutral-buffered formalin. Feet were preserved with the tissues for positive identification of the rat.
At the interima and final sacrifices, the following organs were weighed: brain, heart, liver, spleen, kidneys, testes with epididymides, and ovaries. Adrenals and thyroids with parathyroids were weighed at the final sacrifice. For paired organs, the organ weight was the combined weight of right and left members of the pair. Organ/ body weight ratios were determined for each tissue. No organ weights were taken on rats found dead.
HISTOPATHOLOGY: Yes (see table 1)
The full tissue microscopic examination listed in table 1 was done on the control and high-dose rats and on those dying on study or sacrificed in extremis. Also livers, hearts, and kidneys of low- and mid-dose rats, and all gross lesions seen at necropsy were examined microscopically.

Other examinations:

No further data

Statistics:

The body weight, food consumption, clinicopathologic, and organ weight data were tested for homogeneity of variance by Bartlett's method. If the data were found to be homogeneous, differences between control and treatment means were tested for statistical significance by the method of Dunnett. If the data were found not to be homogeneous, the method of Gill (modified Dunnett's) was employed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were restricted to the high-dose group and included hypoactivity ataxia and salivation.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment related clinical signs were restricted to the high-dose group and included hypoactivity ataxia and salivation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During week 1 the males of the high-dose group gained 18% less than the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For the first 2 weeks of the study, food consumption averages of the high-dose group, males and females were significantly lower than controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment related clinical signs were restricted to the high-dose group and included hypoactivity ataxia and salivation.

BODY WEIGHT AND WEIGHT GAIN
During week 1 the males of the high-dose group gained 18% less than the controls.

FOOD CONSUMPTION
For the first 2 weeks of the study, food consumption averages of the high-dose group, males and females were significantly lower than controls.

OPHTHALMOSCOPIC EXAMINATION
No test compound related effects.

HAEMATOLOGY
No test compound related effects.

CLINICAL CHEMISTRY
No test compound related effects.

URINALYSIS
No test compound related effects.

ORGAN WEIGHTS
No test compound related effects.

GROSS PATHOLOGY
No test compound related effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test compound related effects.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No test compound related effects.

Dose descriptor:

NOAEL

Effect level:

ca. 316 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs; body weight; food consumption

Critical effects observed:

not specified

Lesions were seen in the tissues from the thoracic cavity of all 15 rats which were found dead but gross and microscopic evaluation indicated that these deaths were due either to oesophageal perforation or to tracheal inflammation (and/or necrosis) probably resulting from aspiration of isobutyl alcohol and/or its vapours.

Conclusions:

Oral administration of isobutyl alcohol produced treatment related effects at the highest dose level of 1000 mg/ kg/ day, but not at dose levels of 100 or 316 mg/kg/day. Therefore the NOAEL is considered to be 316 mg/kg/day.

Executive summary:

Groups of male and female rats were dosed orally once daily with 0, 100, 316, or 1000 mg/kg/day of the compound until the day of necropsy at an interim or final sacrifice (days 29 and 30 or days 92 and 93 respectively). The first 10 male and 10 female rats from each group were scheduled for the interim necropsy and all remaining rats were sacrificed on day 92 or 93.

Body weights, food consumption, mortality, any overt signs of toxicity and ophthalmologic examinations were carried out on all animals. Blood and urine were collected from a fifth group of 10 rats/sex prior to initiation of dosing, from all surviving rats at the interim sacrifice and from 10 rats/sex/group at the final sacrifice for clinicopathologic evaluation.

Gross postmortem examinations were done on all rats and organ weights were obtained at the interim and final sacrifices. A histopathologic examination was done on all rats in the control and high-dose groups, on rats found dead or sacrificed moribund, on the livers, hearts, and kidneys of low- and mid-dose rats, and on all gross lesions.

Isobutyl alcohol produced definite treatment-related effects at a dose level of 1000 mg/kg/day, but not at 100 or 316 mg/kg/day. The most pronounced clinical effect was hypoactivity, which was observed during week 1 in every rat at the 1000 mg/kg/day dose level. The incidence of hypoactivity decreased markedly after week 4. During week 1, the body weight gain of the 1000 mg/kg/day males was slightly (18%) lower than that of the control s. Food consumption averages for males and females at this dose level were below control averages for weeks 1 and 2. Serum potassium levels in the 1000 mg/kg/day males and females were 11-15% below the controls of the interim evaluation but similar to controls at the final evaluation.

No effect related directly to treatment was observed in organ weight averages, at the opthalmoscopic examination or at the gross and microscopic tissue examinations. Several rats died during the study, but gross and microscopic evaluation indicated that these deaths were due either to oesophageal perforation or to tracheal inflammation (and/or necrosis) probably resulting from aspiration of isobutyl alcohol and/or its vapours.

In conclusion, the NOAEL was considered to be 316 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

316 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch score = 1. The key study is an acceptable, well documented 90-day repeated dose study which meets scientific principles and is therefore reliable for the determination of the sub-chronic endpoint.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies on the repeated oral dose toxicity of the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate are available. Read-across from the substance to repeated dose toxicity studies on isobutanol and isobutyl isobutyrate is appropriate to meet the REACH Annex VII-IX data requirements.Read-across is scientifically justified and also enables the REACH requirements to be adequately addressed, while avoiding unnecessary animal testing in accordance with EU Directive 86/609/EEC.

The subchronic repeated oral dose toxicity of isobutanol was investigated in rats in a 90-day oral gavage study (Toxicology Research Laboratories, 1987). In the study, groups of thirty male and female CD rats received isobutanol via oral intubation at dose levels of 0, 100, 316, or 1000 mg/kg bw/day for 90 days. Clinical signs related to treatment at the 1000 mg/kg dose level included hypoactivity, ataxia, and salivation. Clinical signs of hypoactivity and ataxia were resolved by the 4th week of the study. Slight decreases in food consumption and body weight gains were noted in the first two weeks and were restricted to the 1000 mg/kg/day group. There were no changes in organ weights or gross or histopathology at any exposure level. The NOAEL for repeated-dose toxicity was considered to be 316 mg/kg bw/day.

The repeated dose oral toxicity of isobutyl isobutyrate in rats was investigated in an 18-week subchronic oral gavage toxicity study, considered to have a reliability score of 2 (Drakeet al., 1978). In the study, Wistar rats (15/sex/dose level) were dosed at levels of 0, 10, 100, or 1,000 mg/kg bw/day for 7 days/week. Control animals received corn oil alone at 5 ml/kg. In addition, groups of five rats of each sex were given the same doses for either 2 weeks or 6 weeks. The animals were weighed on Day 1, 2, 6 and weekly thereafter. After the final dose, the animals were fasted for 24 hours and killed under barbiturate anesthesia.

Examination of blood and urine and the weights and microscopic structure of a limited range of organs were conducted. Hematology parameters were collected at 3, 6, and 18 weeks into the study. A gross necropsy was performed and brain, heart, liver, spleen, kidney, adrenals, stomach, small intestine, caecum, gonads, pituitary, and thyroid(s) were weighed. Typical organs were collected, fixed in formalin, and processed for histological examination. Urine was collected during weeks 2, 6, and 18 of treatment and examined. Kidney function tests were also performed during weeks 6 and 18.

No deaths or abnormal behaviour was observed in the study. No differences in body weight, food or water consumption or histological findings were noted. The only change in haematological parameters observed was a decrease in hemoglobin in males at 100 and 1000 mg/kg bw/day after two weeks of exposure. These changes were not accompanied by changes in other red blood cell parameters in these groups, and the female animals were not affected. In addition, similar findings were not observed at the 6 and 18-week haematology determinations. At 1000 mg/kg bw/day, there was an increase (14%) in relative spleen weight that coincided with a decrease in relative body weights in males which was not considered significant. The mean terminal body weights of animals in this group were decreased by 7% compared to the control group (not statistically significant but may explain the increased relative spleen weight). Due to the fact that there were no histological changes in this or any other organ, this finding was considered spurious.

Based on these findings, the NOAEL for the subchronic, repeated dose toxicity of isobutyl isobutyrate was determined to be 1000 mg/kg bw/day, i.e. the highest dose tested.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Based on existing datasets and structural and chemical considerations, read-across from the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate to available repeated dose toxicity studies on isobutanol and isobutyl isobutyrate is appropriate to meet the REACH Annex VII-IX data requirements. In a sub-chronic toxicity study using isobutanol in rats, the NOAEL was determined to be 316 mg/kg bw/day based on decreased bodyweight gain and transient hypoactivity and ataxia at the higher dose.

Justification for classification or non-classification

Based on read-across to available data on isobutyl isobutyrate and isobutanol, the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate is not predicted to cause any effects that meet the criteria for classification for systemic or target organ toxicity after repeated exposures.On this basis, the substance does not meet the criteria for classification for repeated dose toxicity according to Directive 67/548/EEC or Regulation 1272/2008/EC.