Reaction mass of Amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide

Endpoint summary

Description of key information

Data on repeat dose toxicity by oral route is available for the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide. A 28 -day repeat dose range finding toxicity study was conducted in rats and produced a NOAEL of 300 mg/kg/day. Subsequently, an OECD 422 repeat dose / reproduction screening study was conducted in rats and produced a NOAEL of >300 mg/kg/day.

Read across from soluble lithium salts (lithium chloride – Trautner 1958, and lithium carbonate – SCC 2012) and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and calcium hydroxide is also provided as additional supporting information.

Based on available data the oral NOAEL for the reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide was determined to be 300 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: other guideline: 96/54/EC, B.7 (oral); OECD 407

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar HSD Brl:WH

Sex:

not specified

Route of administration:

oral: unspecified

Vehicle:

other: 1 % Carboxymethylcellulose (CMC)

Details on oral exposure:

Method of administration: stomach tube

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 30 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 750 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 30 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 750 mg/kg bw/day

Details on results:

Clinical observations:
There were no treatment related death. A significant increased weight gain could be detected in the female animals in the Low Dose group. A diminished weight gain was observed in the male Low Dose group.
No treatment related effects were indicated by the results concerning the functional and behavioural examination.

Laboratory findings:
No clinical-chemical parameter showed dosis related changes.
The following significant different values/concentrations/activities were measured: GOT value diminished in the male Low Dose group, GPT value diminished in the female Medium Dose group, AP value diminished in the male Low Dose group, Urea values diminished in the female Medium Dose group, Na values slightly diminished in the male Medium - and High Dose group.
In the assessment of the haematology-values the following parameter were significant decreased in the male Low Dose group: Haemoglobin-value, haematocrit-value, red blood count-values.

Effects in organs:
The mean relativ values of the female High Dose group were significantly lower than the corresponding Control group.
The absoulte weight of the testes of the male High Dose group was significantly lower as compared to values of the Control group.

Correlated histomorphological findings were not observed.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Remarks on result:

other: original NCD unit is mg/kg/day.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

other: original NCD unit is mg/kg/day.

Critical effects observed:

no

Comments:

The value of 1000 mg/kg/day has to be considered as NOAEL as the observed histopathological effects (organ weight changes from spleen and testes) were of no toxicological relevance.

Conclusions:

Classified as: Not classified

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: other guideline: 96/54/EC, B.7 (oral); OECD 407

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar HSD Brl:WH

Sex:

not specified

Route of administration:

oral: unspecified

Vehicle:

other: 1 % Carboxymethylcellulose (CMC)

Details on oral exposure:

Method of administration: stomach tube

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 30 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 750 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 30 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 750 mg/kg bw/day

Details on results:

Clinical observations:
There were no treatment related death. A significant increased weight gain could be detected in the female animals in the Low Dose group. A diminished weight gain was observed in the male Low Dose group.
No treatment related effects were indicated by the results concerning the functional and behavioural examination.

Laboratory findings:
No clinical-chemical parameter showed dosis related changes.
The following significant different values/concentrations/activities were measured: GOT value diminished in the male Low Dose group, GPT value diminished in the female Medium Dose group, AP value diminished in the
male Low Dose group, Urea values diminished in the female Medium Dose group, Na values slightly diminished in the male Medium - and High Dose group.
In the assessment of the haematology-values the following parameter were significant decreased in the male Low Dose group: Haemoglobin-value, haematocrit-value, red blood count-values.

Effects in organs:
The mean relativ values of the female High Dose group were significantly lower than the corresponding Control group.
The absoulte weight of the testes of the male High Dose group was significantly lower as compared to values of the Control group.

Correlated histomorphological findings were not observed.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Remarks on result:

other: original NCD unit is mg/kg/day.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

other: original NCD unit is mg/kg/day.

Critical effects observed:

no

Comments:

The value of 1000 mg/kg/day has to be considered as NOAEL as the observed histopathological effects (organ weight changes from spleen and testes) were of no toxicological relevance.

Conclusions:

Classified as: Not classified

Reference 3

Endpoint:

repeated dose toxicity: oral

Remarks:

other: statement on chronic exposure

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993, 2002, 2007, 2012

Reliability:

1 (reliable without restriction)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Expert statement

GLP compliance:

not specified

Dose descriptor:

NOAEL

Effect level:

6.43 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: In humans.

Critical effects observed:

not specified

Conclusions:

Based on human data obtained from routine long- term treatment of bipolar disorder with lithium, a NOAEL for long-term oral toxicity of 6.43 mg lithium carbonate/kg bw/ day was calculated.

Executive summary:

In humans, lithium/ lithium carbonate has been used for decades in psychiatric therapy for the treatment of bipolar disorder. In case of long-term treatment, the recommended dose is 450 to 900 mg/day lithium carbonate and corresponding to a desired sustained therapeutic serum concentration of 0.5 to 1.0 mmol lithium/L. Based on experience with long-term application e.g. lithium carbonate for therapy in humans, there is no evidence that lithium is of concern with respect to repeated oral toxicity at medical doses as the ones indicated above.

The effect level (NOAEL) determined for lithium carbonate for repeated dose toxicity by the oral route is based on human data and can be calculated in two ways that complete one another:

One option is based on the therapeutic serum concentrations of 0.5 to 1.0 mmol lithium/L and the extracellular fluid (ECF) volume. Lithium has a large volume of distribution of 0.6 - 0.9 L/kg (42 L – 63 L for a 70 kg adult). It is distributed throughout the body water both extra and intracellularly. Lithium shifts into the intracellular compartments of cells because of its large volume of distribution. Although in long-term use, the intracellular concentration increases, the intracellular concentration is not reflected by the plasma level which measures only the extracellular fluid concentration. Therefore, a desired concentration of 1 mmol/L of lithium is expected to be sustained and reflected in the extracellular fluid (ECF) only and not in the intracellular fluid. Thus, the volume considered is of the ECF only which comprises of plasma, interstitial fluid (spaces between cells) and transcellular fluid (lymph, cerebrospinal fluid, synovial fluid, serous fluid, gastrointestinal secretions) and is typically 15 L (reported in different references to be between 14 – 19 L (for 70 kg adult)). Based on this data the derived NOAEL (considering a lithium concentration of 1mmol/L and an ECF volume of 15 L) is 1.5 mg lithium/kg bw/day equivalent to 7.98 mg lithium carbonate/kg bw/day. This NOAEL value can be considered as a conservative value as it is based on an bioavailable dose in humans after absorption and on a smaller volume than its actual distribution volume.

Another way to calculate NOAEL oral for lithium carbonate is based as well on data taken from the routine long-term treatment of bipolar disorder. Instead of calculating the NOAEL from the therapeutic serum concentration of lithium, the lithium carbonate NOAEL oral can be calculated from the administered oral dose for long-term treatment of bipolar disorder as detailed above: 450 to 900 mg lithium carbonate/day (corresponding to the desired sustained concentrations of 0.5 -1 mmole lithium/L in blood/serum). When dividing the oral doses 450 to 900 mg lithium carbonate/day to 70 kg, the following values are obtained respectively: 6.43 to 12.86 mg lithium carbonate/ kg bw/day or when dividing to 60 kg the following values are obtained respectively: 7.5 to 15 mg lithium carbonate/kg bw/day, representing the optional NOAEL values for lithium carbonate for the oral route.

In both ways of calculation, the values obtained are in same order of magnitude and similar to one another. As a worst–case value, a NOAEL repeated dose toxicity oral of 6.43 mg/kg bw/day was chosen. Further, this value could be used as a starting value for route-to-route extrapolation in calculation of the repeated dose toxicity for the dermal and inhalation routes.

Reference 4

Endpoint:

repeated dose toxicity: oral

Remarks:

other: statement on chronic exposure

Type of information:

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

Adequacy of study:

supporting study

Study period:

1993, 2002, 2007, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Expert statement

GLP compliance:

not specified

Dose descriptor:

NOAEL

Effect level:

6.43 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: In humans.

Critical effects observed:

not specified

Conclusions:

Based on human data obtained from routine long- term treatment of bipolar disorder with lithium, a NOAEL for long-term oral toxicity of 6.43 mg lithium carbonate/kg bw/ day was calculated.

Executive summary:

In humans, lithium/ lithium carbonate has been used for decades in psychiatric therapy for the treatment of bipolar disorder. In case of long-term treatment, the recommended dose is 450 to 900 mg/day lithium carbonate and corresponding to a desired sustained therapeutic serum concentration of 0.5 to 1.0 mmol lithium/L. Based on experience with long-term application e.g. lithium carbonate for therapy in humans, there is no evidence that lithium is of concern with respect to repeated oral toxicity at medical doses as the ones indicated above.

The effect level (NOAEL) determined for lithium carbonate for repeated dose toxicity by the oral route is based on human data and can be calculated in two ways that complete one another:

One option is based on the therapeutic serum concentrations of 0.5 to 1.0 mmol lithium/L and the extracellular fluid (ECF) volume. Lithium has a large volume of distribution of 0.6 - 0.9 L/kg (42 L – 63 L for a 70 kg adult). It is distributed throughout the body water both extra and intracellularly. Lithium shifts into the intracellular compartments of cells because of its large volume of distribution. Although in long-term use, the intracellular concentration increases, the intracellular concentration is not reflected by the plasma level which measures only the extracellular fluid concentration. Therefore, a desired concentration of 1 mmol/L of lithium is expected to be sustained and reflected in the extracellular fluid (ECF) only and not in the intracellular fluid. Thus, the volume considered is of the ECF only which comprises of plasma, interstitial fluid (spaces between cells) and transcellular fluid (lymph, cerebrospinal fluid, synovial fluid, serous fluid, gastrointestinal secretions) and is typically 15 L (reported in different references to be between 14 – 19 L (for 70 kg adult)). Based on this data the derived NOAEL (considering a lithium concentration of 1mmol/L and an ECF volume of 15 L) is 1.5 mg lithium/kg bw/day equivalent to 7.98 mg lithium carbonate/kg bw/day. This NOAEL value can be considered as a conservative value as it is based on an bioavailable dose in humans after absorption and on a smaller volume than its actual distribution volume.

Another way to calculate NOAEL oral for lithium carbonate is based as well on data taken from the routine long-term treatment of bipolar disorder. Instead of calculating the NOAEL from the therapeutic serum concentration of lithium, the lithium carbonate NOAEL oral can be calculated from the administered oral dose for long-term treatment of bipolar disorder as detailed above: 450 to 900 mg lithium carbonate/day (corresponding to the desired sustained concentrations of 0.5 -1 mmole lithium/L in blood/serum). When dividing the oral doses 450 to 900 mg lithium carbonate/day to 70 kg, the following values are obtained respectively: 6.43 to 12.86 mg lithium carbonate/ kg bw/day or when dividing to 60 kg the following values are obtained respectively: 7.5 to 15 mg lithium carbonate/kg bw/day, representing the optional NOAEL values for lithium carbonate for the oral route.

In both ways of calculation, the values obtained are in same order of magnitude and similar to one another. As a worst–case value, a NOAEL repeated dose toxicity oral of 6.43 mg/kg bw/day was chosen. Further, this value could be used as a starting value for route-to-route extrapolation in calculation of the repeated dose toxicity for the dermal and inhalation routes.

Reference 5

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Study period:

1958, 1995, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publications

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Principles of method if other than guideline:

No data

GLP compliance:

not specified

Remarks:

study was published in 1958

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight:
females: 0.14 kg - 0.18 kg
males: 0.2 - 0.27 kg

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

10-12 g/0.1 kg/bw /day

Dose descriptor:

NOAEL

Effect level:

13.9 mg/kg bw/day (nominal)

Based on:

other: conversion LiCl -> Li

Sex:

male

Basis for effect level:

other: Result refers to lithium

Dose descriptor:

NOAEL

Effect level:

73.89 mg/kg bw/day (nominal)

Based on:

other: conversion Li -> Li2CO3

Sex:

male

Basis for effect level:

other: Result refers to lithium carbonate

Critical effects observed:

not specified

Dose level 20 mmol lithium chloride / L:

This dose was just subtoxic; apart from slight initial disturbances, it caused no effects on health or behaviour in the adult animals over a period of up to two years.

Plasma levels: 1.5 - 2 mmol lithium

Dose level 50 mmol lithium chloride / L:

The animals appeared to be unchanged in aspect and behaviour for 3 to 5 days. Then they became progressively drowsy and asocial. They were difficult to rouse; when roused, their gait was staggering and hesitant and they rested again as soon as possible. When resting they showed fine muscular tremor and trembling; usually they sat for long periods by themselves anywhere in the box. A few days later they became unresponsive and were stuporous, with dull eyes and slightly erected, untidy and unkempt fur. They were visibly ill and emaciated. Death occurred within 2 to 3 weeks.

Plasma levels: 3 (behavioural changes) - 8 (death) mmol lithium

Conclusions:

In a 2-year study in rats ingesting drinking water containing lithium chloride in a concentration of 20 mmol no effects on health or behaviour were found, except slight, transitory initial disturbances. When a concentration of 50 mmol LiCl/L was administered, food and water intake was decreased within a few days, and the rats became progressively drowsy and asocial on the 3rd to 5th day. Their gait was staggering, and they had a fine muscular tremor. Simultaneously their weight began to drop. The deterioration progressed to stupor and death within 2-3 weeks.
Plasma levels at dose level 20 mmol/L: 1.5 - 2 mmol
Lithium Plasma levels at dose level 50 mmol/L: 3 (behavioural changes) - 8 (death) mmol lithium.

The lithium concentration of 20 mmol/L used in this chronic drinking water study, is comparable to the highest doses given temporarily to hospitalised patients (plasma lithium level about 2 mmol/L).

The daily water intake was 0.010 - 0.012 kg / 0.1 kg bw.
For worst case considerations, the daily lithium intake in rat is 2.0 mmol/kg bw/day which is equivalent to 13.881 lithium mg/kg bw/ day.
Thus, a worst case NO(A)EL of 13.9 mg lithium/kg bw/ day can be derived.

Executive summary:

In a 2-year study in rats ingesting drinking water containing lithium chloride in a concentration of 20 mmol no effects on health or behaviour were found, except slight, transitory initial disturbances. When a concentration of 50 mmol LiCl/L was administered, food and water intake was decreased within a few days, and the rats became progressively drowsy and asocial on the 3rd to 5th day. Their gait was staggering, and they had a fine muscular tremor. Simultaneously their weight began to drop. The deterioration progressed to stupor and death within 2-3 weeks.

Plasma levels at dose level 20 mmol/L: 1.5 - 2 mmol

Lithium Plasma levels at dose level 50 mmol/L: 3 (behavioural changes) - 8 (death) mmol Lithium.

Based on the result, a worst case NOAEL of 2.0 mmol/kg bw/day, respectively, 13.9 Lithium mg/kg bw/ day can be derived.

Reference 6

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Study period:

1958, 1995, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publications

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Principles of method if other than guideline:

No data

GLP compliance:

not specified

Remarks:

study was published in 1958

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight:
females: 0.14 kg - 0.18 kg
males: 0.2 - 0.27 kg

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

10-12 g/0.1 kg/bw /day

Dose descriptor:

NOAEL

Effect level:

13.9 mg/kg bw/day (nominal)

Based on:

other: conversion LiCl -> Li

Sex:

male

Basis for effect level:

other: Result refers to lithium

Dose descriptor:

NOAEL

Effect level:

73.89 mg/kg bw/day (nominal)

Based on:

other: conversion Li -> Li2CO3

Sex:

male

Basis for effect level:

other: Result refers to lithium carbonate

Critical effects observed:

not specified

Dose level 20 mmol lithium chloride / L:

This dose was just subtoxic; apart from slight initial disturbances, it caused no effects on health or behaviour in the adult animals over a period of up to two years.

Plasma levels: 1.5 - 2 mmol lithium

Dose level 50 mmol lithium chloride / L:

The animals appeared to be unchanged in aspect and behaviour for 3 to 5 days. Then they became progressively drowsy and asocial. They were difficult to rouse; when roused, their gait was staggering and hesitant and they rested again as soon as possible. When resting they showed fine muscular tremor and trembling; usually they sat for long periods by themselves anywhere in the box. A few days later they became unresponsive and were stuporous, with dull eyes and slightly erected, untidy and unkempt fur. They were visibly ill and emaciated. Death occurred within 2 to 3 weeks.

Plasma levels: 3 (behavioural changes) - 8 (death) mmol lithium

Conclusions:

In a 2-year study in rats ingesting drinking water containing lithium chloride in a concentration of 20 mmol no effects on health or behaviour were found, except slight, transitory initial disturbances. When a concentration of 50 mmol LiCl/L was administered, food and water intake was decreased within a few days, and the rats became progressively drowsy and asocial on the 3rd to 5th day. Their gait was staggering, and they had a fine muscular tremor. Simultaneously their weight began to drop. The deterioration progressed to stupor and death within 2-3 weeks.
Plasma levels at dose level 20 mmol/L: 1.5 - 2 mmol
Lithium Plasma levels at dose level 50 mmol/L: 3 (behavioural changes) - 8 (death) mmol lithium.

The lithium concentration of 20 mmol/L used in this chronic drinking water study, is comparable to the highest doses given temporarily to hospitalised patients (plasma lithium level about 2 mmol/L).

The daily water intake was 0.010 - 0.012 kg / 0.1 kg bw.
For worst case considerations, the daily lithium intake in rat is 2.0 mmol/kg bw/day which is equivalent to 13.881 lithium mg/kg bw/ day.
Thus, a worst case NO(A)EL of 13.9 mg lithium/kg bw/ day can be derived.

Executive summary:

In a 2-year study in rats ingesting drinking water containing lithium chloride in a concentration of 20 mmol no effects on health or behaviour were found, except slight, transitory initial disturbances. When a concentration of 50 mmol LiCl/L was administered, food and water intake was decreased within a few days, and the rats became progressively drowsy and asocial on the 3rd to 5th day. Their gait was staggering, and they had a fine muscular tremor. Simultaneously their weight began to drop. The deterioration progressed to stupor and death within 2-3 weeks.

Plasma levels at dose level 20 mmol/L: 1.5 - 2 mmol

Lithium Plasma levels at dose level 50 mmol/L: 3 (behavioural changes) - 8 (death) mmol Lithium.

Based on the result, a worst case NOAEL of 2.0 mmol/kg bw/day, respectively, 13.9 Lithium mg/kg bw/ day can be derived.

Reference 7

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2019 to August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 78 - 84 days (males), 71 - 77 days (females)
- Weight at study initiation: Males: 336 - 454 g; Females: 202 - 277 g;
- Housing: House 2 or 3 per cage by sex in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 23 - 66
- Air changes (per hr): 10 minimum
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27 March 2019 To: 25 April 2019 (males) and 15-21 May 2019 (females and pups)

Route of administration:

oral: gavage

Vehicle:

other: Propylene glycol 10% in Milli-Q Water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared as required and transferred to the animal unit immediately or stored in a refrigerator set to maintain 4°C, protected from light and dispensed daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by Ultra Performance Liquid Chromatography (UPLC) using a validated analytical procedure.

Duplicate sets of top, middle and bottom samples (duplicate middle only for control) were collected for analysis; triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. Samples volumes were collected as follows: Groups 1 and 2: 0.2 mL into a 5 mL volumetric flask and Groups 3 and 4: 0.1 mL into a 10 mL volumetric flask. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of 10% for each group.

Duration of treatment / exposure:

Males: Males were dosed once daily for 29 days, starting from 14 days prior to mating.
Females: Females were dosed once daily from 14 days prior to mating, then continuing through gestation and lactation periods until the day prior to termination (LD 13). In this way female animals were dosed from Study Day 1 up to Day 54 (dependent on mating).

Frequency of treatment:

Daily.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the findings from two previous preliminary range-finding studies conducted in male and female (non-pregnant) rats.
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were subjected to detailed clinical observations weekly from Week -1.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once during pretreatment and daily during the dosing period. Body weight for non-pregnant animals are not reported during lactation (not collected).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day.
- Time schedule for examinations: Food consumption was quantitively measured weekly for both sexes from Week -1 until pairing for mating, and on GD 0 to 7, 7 to 14 and 14 to 20 and LD 1 to 7 and 7 to 13 for the mated females, where possible.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

OTHER: Ophthalmic Examinations
- Time schedule for examinations: Animals were subject to ophthalmic examinations once during pre-treatment (all animals) and once during Week 4 (males only) or the week before termination (females only).

OTHER: Detailed functional observations
- Time schedule for examinations: Detailed functional observations were conducted for all animals once during pretreatment and weekly throughout the dosing period for all adult animals. From Week 6 onwards, only selected females continued to have detailed functional observations.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals on test day 30
- Maternal animals: All surviving animals on lactation day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights: Brain, epididymis, adrenal glands, pituitary glands, prostate, thyroid/parathyroid glands, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus.
- Microscopic evaluation: Bone marrow, brain, cervix, epididymis, oesophagus, eye, adrenal glands, harderian gland, mammary gland, parathyroid glands, pituitary glands, prostate, salivary glands, seminal vesicles, thyroid glands, gut-associated lymphoid tissue, heart, femorotibial joint, kidney, colon, cecum, rectum, liver, lung, lymph nodes, optic nerve, sciatic nerve, ovaries, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, tongue, trachea, ureter, urinary bladder, uterus, vagina.

Statistics:

Levene’s test was used to assess the homogeneity of group variances.
Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animal 4003M at 300 mg/kg/day displayed cranial twitches prior to dosing on Day 22. The animal was still considered fit for dosing and there were no further occurrences.

In addition, at 300 mg/kg/day sporadic observations were indicative of potential toxicity, including: irregular respiration rate, hunched posture (also observed in one occasion at 75 mg/kg/day), cold to touch, piloerection and abnormal gait. Due to the short duration and infrequent occurrence these observations were considered to be not adverse.

Ploughing behaviour and salivation (including wet fur on lower jaw), whilst most prominent at 300 mg/kg/day (and in particular in females during gestation), were considered to likely be response to dosing and not adverse or evidence of toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight or body weight gains in male or female animals over the course of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no appreciable effect on the food consumption of male animals. In female animals prior to mating a difference was observed between the food consumption at 150 mg/kg/day animals over Day 8-15 compared with both the control (14.5 g/animal/day verses 19.3 g/animal/day), and compared with the pre-dose values (17.8 g/animal/day). As this effect was not observed at 300 mg/kg/day it is considered unlikely to be associated with the administration of the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were test item changes in the ophthalmic observations following administration of the test item.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item related changes to haematology parameters in this study. There were no test item related changes to coagulation parameters in this study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a dose-related change in the concentration of circulating sodium levels, and whilst statistically significant, were within a typical rage for rats and were considered not adverse.

Other parameters attained statistical significance but did not follow a dose-related pattern and were considered spurious and not related to the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in the functional observations and behaviour following the administration of the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related organ weight differences.

There were additional organ weight differences, which were considered not to be test item related: In males, there were higher thyroid weights at 150 and 300 mg/kg/day, and higher liver weights at
75 and 300 mg/kg/day. In females, there were higher brain weights at 150 and 300 mg/kg/day. However, there was no dose response for any of these weight changes. There were higher spleen weights at 300 mg/kg/day, for which there was no microscopic correlate. There were individual organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related gross findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related microscopic findings.

The only microscopic finding of note was in males and consisted of an increased incidence of splenic haemopoiesis at 300 mg/kg/day which was considered not to be test item-related since there were no correlating haematology findings.
Other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Stimulating Hormone (TSH) and Thyroxine (T4):
In male animals (F0) lower levels of TSH were observed at 300 mg/kg/day (2.03 ng/mL at 300 mg/kg/day compared with 3.13 ng/ml in the male control animals). There was also a higher incidence of animals with concentrations of TSH Female animals (F0) were found with a lower level of TSH, a modest reduction was noted at 300 mg/kg/day (1.35 ng/mL compared with 1.64 ng/mL in the female control animals). There were considered to be no changes to concentrations of T4 attributed to the test item.
These micro changes in circulating thyroid hormone levels are considered to be of doubtful toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Not determinable

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

In conclusion, administration of Lithium-Azelate-Thickener by once daily oral gavage was well tolerated in Sprague Dawley (CD) rats. There was no evidence of test item toxicity. The No Observed Effect Level (NOEL) was considered to be 150 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was 300 mg/kg/day in this study.

Executive summary:

A study to assess the toxicity of the substance in the rat after repeated oral administration and to provide initial information on possible effects on reproduction and/or development and neurotoxicity was conducted according to OECD 422 guideline. Three test groups and one control group, each containing 10 males and 10 females were used in the study. Males were treated for 2 weeks prior to mating until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation. The dose levels for the study were based on the findings from two previous preliminary range-finding studies conducted in male and female (nonpregnant) rats and were selected to be 75, 150 and 300 mg/kg/day where the high dose level was expected to induce minimal tolerated toxicity in the form of slight body weight effects in the parent animals and the low dose levels would be a no-effect level in the parents. No overt toxicity was produced in the rats at the dose levels tested. The no observed effect level (NOEL) was considered to be 150 mg/kg/day and the no observed adverse effect level was 300 mg/kg/day.

Reference 8

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2018 to 23 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Dose range-finding studies to assess the toxicity of Lithium-Azelate-Amine thickener

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for toxicity testing and the number of animals used was considered the minimum to properly assess the potential effects.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent
- Age at study initiation: Phase 1 - 18 to 19 weeks old; Phase 2 - 10 to 12 weeks old
- Weight at study initiation: Phase 1 - 245 to 593 grams; Phase 2 - 416 to 564 grams
- Fasting period before study: Not reported
- Housing: Animals were housed 2 or 3 per cage in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms
- Diet (e.g. ad libitum): SDS VRF-1 breeder diet provided ad libitum
- Water (e.g. ad libitum): Water provided ad libitum
- Acclimation period: Phase 1 - 13 weeks; Phase 2 - 28 to 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 3 - 23 °C
- Humidity (%): 5 - 137%
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Route of administration:

oral: gavage

Details on route of administration:

Doses were administered orally using plastic gavages. The volume administered to each animal was determined on each day by the weight of each animal on the day.

Vehicle:

other: 10% Propylene Glycol in Milli-Q Water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analytical verification of doses

Duration of treatment / exposure:

Phase 1 - 4 days
Phase 2 - 21 days

Frequency of treatment:

Daily

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Phase 1 - administered on test days 1 to 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Phase 1 - administered on test days 1 to 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Phase 2 - administered on test days 1 - 21

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Phase 2 - administered on test days 1 - 21

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Phase 2 - administered on test days 3 - 21

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Phase 2 - administered on test days 6 - 21

No. of animals per sex per dose:

Phase 1 - 2 males and 3 females (1000 mg/kg/day) and 3 males and 2 females (600 mg/kg/day)

Phase 2 - 10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed prior to dosing and regularly throughout the day on each day of dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were subject to detailed clinical observations weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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): Yes
- Time schedule for examinations: Monitored on a regular basis

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Details: All phase 1 and phase 2 animals subject to gross pathology

ORGAN WEIGHTS:
Phase 1: Liver
Phase 2: Brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus, uterus.

HISTOPATHOLOGY:
Not performed.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 0.1%, 1%, and 5% levels.

The following pairwise comparisons were made:
Group 4 vs. Group 3
Group 5 vs. Group 3
Group 6 vs. Group 3

Levene’s test was used to assess the homogeneity of group variances.

Datasets with at least 3 groups were compared using an overall one-way ANOVA F-test when Levene’s test was not significant or the Kruskal-Wallis test when it was. When the overall F-test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Clinical signs:

no effects observed

Description (incidence and severity):

Phase 1: Lithium-azelate-amine thickener administered to rats at 600 or 1000 mg/kg/day in the Dose Level Confirmation phase induced excessive toxicity. Clinical signs of piloerection, decreased activity and eyes partly closed were observed in the first few days of dosing. Male animals dosed at 1000 mg/kg/day and all female animals displayed the clinical sign of abnormal gait.

Phase 2: Administration of Lithium-azelate-amine thickener at dose levels of 100, 200 or 300 mg/kg/day was well tolerated. Clinical observations of fur staining were considered incidental and did not follow a treatment related pattern.

Mortality:

mortality observed, treatment-related

Description (incidence):

Phase 1: Excessive mortality was observed in the Dose Level Confirmation phase. There was no evidence of a recovery on Day 4 to Day 5. Animals were humanly killed on Day 5.

Phase 2: There were no unscheduled deaths in Groups 3-6 (the repeat dose phase).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Phase 1: Substantial body weight losses were observed in both groups (approximately 10% bodyweight loss in 5 days).

Phase 2: During the first week of dosing (Days 1-8) the body weights gains in male rats were lower at 200 and 300 mg/kg/day, with mean body weight gains of 20.7 g and 14.9 g respectively compared with 30.5 g in the control. During the second week of dosing, the bodyweight gains in male rats at 300 mg/kg/day were also lower than control (14.5 g compared with 19.2 g in the control).
In female rats bodyweight gains were lower at 300 mg/kg/day, with mean body weight gains of 7.1 g compared with 13.2 g in the control between Day 1-8, and 8.2 g compared with 14.3 g between Day 8-15.
Administration of Lithium-azelate-amine thickener at dose levels of 100 or 200 mg/kg/day in female or 100 mg/kg/day in male rats did not affect body weight or body weight gains.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Phase 1: Administration of Lithium-azelate-amine thickener at both the 600 and the 1000 mg/kg/day dose level caused strong suppression of food consumption. In males between Day 1 and Day 4 of dosing, food consumption was calculated to be 8.3 and 5.7 g/animal/day at 1000 and 600 mg/kg/day, respectively. In females at this time food consumption was <1 g/animal/day.

Phase 2: In male animals, administration of Lithium-azelate-amine thickener was not associated with a change in food consumption. In female animals, administration of Lithium-azelate-amine thickener at 300 mg/kg/day produced a modestly lower food consumption was seen between Days 1-3 of dosing compared with pre-treatment values (14 -15 g/animal/day compared with 22 g pre-treatment). These values were also lower than the control animals throughout the study. From Day 4 of dosing food consumption returned to a typical level (i.e. similar to the pretreatment). There were no evident differences in food consumption at 200 or 100 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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):

Phase 1: Not examined.

Phase 2: In the male animals, there was a lower absolute and relative thymus weights in all test item groups administered with Lithium-azelate-amine thickener compared with the control. In addition, the absolute adrenal gland weight was higher at 300 mg/kg/day. When comparing the weights relative to body weights, the relative adrenal gland weights were higher in all groups administered with Lithium-azelate-amine thickener than the control. Detailed organ weight differences provided in Table 1.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Phase 1: In Groups 1 and 2, during the necropsy it was observed that the stomach contents of animals were abnormal: white/thick/gelatinous (all animals at 1000 -with the exception of 1002- or 600 mg/kg/day). It is considered likely that this was due to the dosing formulations / test item not passing through the stomach. Other findings were suggestive that there could be a potential irritation to the GI tract, this included: dark, red discolouration of the rectum; the ileum and of the stomach (glandular and non-gladular). Promenent lobular architecture of the liver was also noted.

Phase 2: Necropsy examination indicated some potential liver differences in male animals, with an increase incidence of the finding of prominent lobular architecture compared with absence in controls. The finding of abnormal stomach contents (white/thick) in one control animal was considered spurious. There was no evidence in the test item groups that the formulations were not being processed through the GI tract of the animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Not determinable

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

In the dose level confirmation phase, dose levels of 1000 or 600 mg/kg/day produced reduced food consumption, leading to bodyweight losses and clinical signs of poor condition. Dosing was terminated however there were no improvements in the condition of the animals, and they were killed prematurely. Necropsy examination revealed findings of white/thick/gelatinous material in the stomach believed to be the test item formulation. There was also evidence of discolouration in the gastrointestinal tract.

Dose levels were lowered in Phase 2. In this phase there was a reduction in bodyweight gains in males at 200 and 300 mg/kg/day and females at 300 mg/kg/day. Food consumption was also lower in females at 300 mg/kg/day at the start of the dosing period. Organ weight differences were noted. Increased adrenal weights in males at 300 mg/kg/day, decreased uterus weights in females at 300 mg/kg/day, decreased pituitary weights in females at 300 mg/kg/day and decreased thymus weight in males and females at 300 mg/kg/day.

Table 1 - Phase 2 organ weight differences

	Absolute				Relative (to body)
Organ	4M	5M	6M	6F	4M	5M	6M	6F
Adrenal gland	-	-	+11%	-	+12%	+10%	+18%	-
Thymus	-21%	-16%	-18%	-15%	-19%	-12%	-14%	-12%
Pituitary gland	-	-	-	-17%	-	-	-	-16%
Uterus	-	-	-	-18%	-	-	-	-15%

Differences compared to controls.

Conclusions:

In conclusion, administration of lithium-azelate-amine thickener by repeat-dose, once daily oral gavage was not tolerated at 1000 or 600 mg/kg/day in Phase1 of the study due to clinical signs and sustained low or absent food consumption. In Phase 2, administration at up to 300 mg/kg/day was tolerated for 16 days and was associated with lower body weight gain (both sexes) and transiently lower food consumption (females only). An increase in the adrenal weight and reduction in thymus weight (males only), uterus (females only) and pituitary (females only) were evident, however were considered not adverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Executive summary:

A preliminary range-finding study was conducted to assess the toxicity of the substance Lithium-Azelate-Amine Thickener in the rat after oral gavage administration up to 21 days. Initially, a dose level confirmation phase consisting of 10 animals examined the acute oral tolerance of the test item up to the limit dose. Five animals (2 males and 3 females) were treated with 1000 mg/kg/day for 4 days (Group 1) and another five animals (3 males and 2 females) were treated with 600 mg/kg/day for 4 days (Group 2). During the second phase, three test groups and one control group, each containing 10 males and 10 females, were used to more fully determine the toxicology of the test item. Group 3 control animals were treated with 0 mg/kg/day for 21 days. Group 4 were treated with 100 mg/kg/day for 21 days, group 5 were treated with 200 mg/kg/day for 19 days (TD 3-21) and group 6 were treated with 300 mg/kg/day for 16 days (TD 6-21). In the dose level confirmation phase, dose levels of 1000 or 600 mg/kg/day produced reduced food consumption, leading to bodyweight losses and clinical signs of poor condition. Dosing was terminated however there were no improvements in the condition of the animals, and they were killed prematurely. Necropsy examination revealed findings of white/thick/gelatinous material in the stomach believed to be the test item formulation. There was also evidence of discolouration in the gastrointestinal tract.

Dose levels were lowered in Phase 2. In this phase there was a reduction in bodyweight gains in males at 200 and 300 mg/kg/day and females at 300 mg/kg/day. Food consumption was also lower in females at 300 mg/kg/day at the start of the dosing period. Organ weight differences were noted. Increased adrenal weights in males at 300 mg/kg/day, decreased uterus weights in females at 300 mg/kg/day, decreased pituitary weights in females at 300 mg/kg/day and decreased thymus weight in males and females at 300 mg/kg/day. However, these results were considered non-adverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 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

Repeated oral toxicity

Data on repeat dose toxicity by oral route is available for the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide. A 28 -day repeat dose range finding toxicity study was conducted in rats and produced a NOAEL of 300 mg/kg/day. A study to assess the toxicity of the substance in the rat after oral administration and to provide initial information on possible effects on reproduction and/or development and neurotoxicity was conducted according to OECD 422 guideline (Bain 2019).Three test groups and one control group, each containing 10 males and 10 females were used in the study. Males were treated for 2 weeks prior to mating until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation and lactation until LD13. The dose levels selected for use in this study were based on the findings from two previous preliminary range-finding studies conducted in male and female (non-pregnant) rats. The test material was well tolerated in Sprague Dawley (CD) rats. There was no evidence of test item toxicity. The No Observed Effect Level (NOEL) was considered to be 150 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was 300 mg/kg in this study.

Read across from soluble inorganic lithium salts and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and calcium hydroxide is also provided as additional supporting information.

In humans, lithium/lithium carbonate has been used for decades in psychiatric therapy for the treatment of bipolar disorder. In case of long-term treatment, the recommended dose is 450 to 900 mg/day lithium carbonate and corresponding to a desired sustained therapeutic serum concentration of 0.5 to 1.0 mmol lithium/L. Based on experience with long-term application e.g. lithium carbonate for therapy in humans, there is no evidence that lithium is of concern with respect to repeated oral toxicity at medical doses as the ones indicated above.

The effect level (NOAEL) determined for lithium carbonate for repeated dose toxicity by the oral route is based on human data and can be calculated in two ways that compliment one another:

One option is based on the therapeutic serum concentrations of 0.5 to 1.0 mmol lithium/L and the extracellular fluid (ECF) volume. Lithium has a large volume of distribution of 0.6 - 0.9 L/kg (42 L – 63 L for a 70 kg adult). It is distributed throughout the body water both extra and intracellularly. Lithium shifts into the intracellular compartments of cells because of its large volume of distribution. Although in long-term use, the intracellular concentration increases, the intracellular concentration is not reflected by the plasma level which measures only the extracellular fluid concentration. Therefore, a desired concentration of 1 mmol/L of lithium is expected to be sustained and reflected in the extracellular fluid (ECF) only and not in the intracellular fluid. Thus, the volume considered is of the ECF only which comprises plasma, interstitial fluid (spaces between cells) and transcellular fluid (lymph, cerebrospinal fluid, synovial fluid, serous fluid, gastrointestinal secretions) and is typically 15 L (reported in different references to be between 14 – 19 L (for 70 kg adult)). Based on this data the derived NOAEL (considering a lithium concentration of 1mmol/L and an ECF volume of 15 L) is 1.5 mg lithium/kg bw/day equivalent to 7.98 mg lithium carbonate/kg bw/day. This NOAEL value can be considered as a conservative value as it is based on a bioavailable dose in humans after absorption and on a smaller volume than its actual distribution volume.

Another way to calculate NOAEL oral for lithium carbonate is based on data taken from the routine long-term treatment of bipolar disorder. Instead of calculating the NOAEL from the therapeutic serum concentration of lithium, the lithium carbonate NOAEL oral can be calculated from the administered oral dose for long-term treatment of bipolar disorder as detailed above: 450 to 900 mg lithium carbonate/day (corresponding to the desired sustained concentrations of 0.5 -1 mmole lithium/L in blood/serum). When calculating the oral doses 450 to 900 mg lithium carbonate/day to a 70 kg person, the following values are obtained respectively: 6.43 to 12.86 mg lithium carbonate/ kg bw/day or when calculating for a 60 kg person the following values are obtained respectively: 7.5 to 15 mg lithium carbonate/kg bw/day, representing the optional NOAEL values for lithium carbonate for the oral route.

In both ways of calculation, the values obtained are in same order of magnitude and similar to one another. As a worst–case value, a NOAEL repeated dose toxicity oral of 6.43 mg/kg bw/day was chosen. Further, this value could be used as a starting value for route-to-route extrapolation in calculation of the repeated dose toxicity for the dermal and inhalation routes.

In another weight of evidence (Trautner, 1958), a 2-year study in rats with lithium chloride administered in drinking water, resulted in a NOAEL of 13.9 mg lithium/kg bw/day. This result is higher than the human NOAEL derived (1.2 mg lithium/kg bw/day). Giving preference to human data, to worst-case result, and to the most reliable data the NOAEL value determined is 1.2 mg lithium/kg bw/day corresponding to 6.43 mg lithium carbonate/kg bw/day.

A sub-acute oral gavage toxicity study on male and female Wistar rats was conducted according to the OECD 407 guideline with reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and calcium hydroxide (BIOSERVICE SCIENTIFIC LABORATORIES,1999). The study gave a NOAEL of 1000 mg/kg bw/day.

Justification for classification or non-classification

In a 28 -day oral toxicity range finding study and an OECD 422 repeat dose / reproduction screening test, the NOAEL was determined to be 300 mg/kg/day in male and female rats.

Conclusion: Not classified for STOT systemic. No toxicologically significant adverse effects observed.