Betaines, C12-14 (even numbered)-alkyldimethyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

Reference to dose range finding study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

2 week preliminary oral toxicity study in rats

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 2020 - January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is a 14-day dose range finding study for the OECD 408 study, therefore it is only supportive

Justification for type of information:

The purpose of this study was to investigate the toxicity ofBetaines, C12-14 (even numbered)-alkyldimethyl in rats after daily oral administration for 2 weeks, in order to find suitable dose levels for the OECD 408 study on this substance.

Reason / purpose for cross-reference:

reference to other study

Remarks:

Preliminary dose range finding study for this referenced OECD 408 study.

Qualifier:

no guideline required

Principles of method if other than guideline:

This study was a preliminary dose range finding study for the OECD 408 study. The toxicity of Betaines, C12-14 (even numbered)-alkyldimethyl was investigated in rats after daily oral administration for 2 consecutive weeks.

GLP compliance:

no

Remarks:

This study was a preliminary dose range finding study for the OECD 408 study and is exempt from compliance with the Principles on Good Laboratory Practice of the OECD. However, it will be carried out in a GLP compliant facility.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Source: Innospec Limited
Identity: Betaines, C12-14 (even numbered)-alkyldimethyl
Label name: EMPIGEN® BB/FL
EC number: 931-700-2
Batch no: BLBX030618
Retest date: 04 January 2022
Appearance: Clear liquid
Purity: 29.92%
Correction factor 3.34

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition of test material: Room temperature, protected from light and moisture
Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: preparation was made at weekly intervals at concentrations of 1, 3 and 10 mg/mL and stored at room temperature for a maximum of 8 days, based on results from ERBC Study no. A4171 (28 hour and an 8 day stability at room temperature were verified in the range from 1 to 100 mg/mL).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Treatment of test material prior to testing: N/A

OTHER SPECIFICS
C-Chain analysis of batch BLBX030618 (Subs ID Report RT0158S) show that material fits the REACH substance Identity profile.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD 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 B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 27-29 days old
- Weight at study initiation: 111.9-112.3g for males and 110.8-111.7g for females
- Housing: The animals were housed in a limited access rodent facility. The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water (e.g. ad libitum): ad libitum to each cage via water bottles
- Acclimation period: 12 days

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. Records of analysis of water, diet and bedding material are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

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

On the day of allocation (6 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 4 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group will be evenly distributed across the battery to minimise possible environmental effects.

IN-LIFE DATES:
Start of experimental phase (ERBC) - Allocation of animals to the study: 16 December 2021
End of experimental phase (ERBC) - Last day of necropsy: 05 January 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:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The preparations were made daily (concentrations of 3, 12.5 and 50mg/mL). Concentrations were calculated and expressed in terms of test item corrected for purity (correction factor 3.34).

VEHICLE
- Vehicle: softened water (by reverse osmosis)
- Concentration in vehicle: 3, 12.5 and 50 mg/mL for low-, mid- and high-dose group, respectively.

ADMINISTRATION OF TEST ITEM:
The test item was administered orally, by gavage at a dose volume of 10mL/kg. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

All animals were dosed once a day at a similar time each day, 7 days a week, for 2 consecutive weeks, until the day before necropsy.

Analytical verification of doses or concentrations:

no

Remarks:

Not necessary for dose range finding experiment

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily, 7 days / week

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Each group comprised 4 male and 4 female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

The toxicity of Betaines, C12-14 (even numbered)-alkyldimethyl was investigated in rats after daily oral administration for 2 weeks. Three groups, each of 4 male and 4 female Sprague Dawley SD rats, received the test item once daily by gavage, at dose levels of 30, 125 and 500 mg/kg/day for 2 consecutive weeks. A fourth, similarly constituted group received the vehicle alone (softened water) and acted as control.

The animals were observed for mortality, clinical signs, body weight and food consumption. At termination, all animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention. Microscopic evaluation of the stomach was performed in all animals from Group 1 (control) and those treated at 125 and 500 mg/kg/day (Groups 3 and 4, respectively).

Dose selection rationale: Doses for the range finder were based on available repeat dose data for structural analogues of C12-14 ADB, in these studies irritation at the sites of contact (the forestomach and bladder) was consistently seen at doses above 150 mg/kg bw/day.

Fasting period before blood sampling for clinical biochemistry: N/A

Post-exposure recovery period in satellite groups: N/A

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality: Throughout the study, all animals were checked early in each working day and again in the afternoon for mortality. At weekends and Public Holiday, a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
Clinical signs: All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day (0.5/1 h post-dose).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

Terminal studies
Euthanasia: Animals in extremis and those that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals, including those found dead, were subjected to necropsy, supervised by a pathologist, as detailed below.

Necropsy: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative.

Organ weights: From all animals completing the scheduled test period, the organs indicated in Attachment 1, Annex 1 under section "Attached background material” were dissected free of fat and weighed. The ratios of organ weight to body weight and brain weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in Attachment 1, Annex 1 under section "Attached background material” were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
Histopathological examination of the stomach was performed for all animals from Group 1 (control) and those treated at 125 and 500 mg/kg/day (Groups 3 and 4, respectively).

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs, mainly including hunched posture, piloerection, and/or swollen abdomen, and/or rales, and salivation occurred in all males and surviving females of the high dose group (500 mg/kg/day), throughout the study. Dyspnoea and decreased activity were also observed in all females dosed at 500 mg/kg/day. On occasion, hunched posture was observed in 3 females (nos. E0575017, E0575019 and E0575021) from mid-dose group (125 mg/kg/day) on Days 3 and 4.

For details on clinical signs, please refer to Attachment 2 - DRF Summary table 1 under section "Attached background material".

Mortality:

mortality observed, treatment-related

Description (incidence):

Premature death occurred in three treated females: no. E0575023 of mid-dose group (125 mg/kg/day), and nos. E0575025 and E0575029 of high dose group (500mg/kg/day). Females nos. E0575023 and E0575025 were found dead on Day 3 and 5 of dosing, respectively, whereas female no. E0575029 was humanely sacrificed on Day 5 of dosing. Animal no. E0575023 (125 mg/kg/day): no clinical signs were observed in this animal. At macroscopic examination, dark red colour of thymus and dark red fluid in the thoracic cavity were reported. Microscopically, oedema and minimal alveolar macrophage aggregations were noted. The macro and microscopic findings were considered due to misgavage. Animal no. E0575025 (500 mg/kg/day): ataxia, decreased activity, dyspnoea, hunched posture and piloerection were recorded in this female during the days prior to death. Macroscopic observations revealed red colour of brain, stomach (glandular and non glandular region) and thymus. At the histopathological evaluation the stomach of animal no. E0575025 was autolytic and therefore not evaluable. Animal no. E0575029 (500 mg/kg/day): decreased activity, dyspnoea, emaciation, hunched posture, matted fur, pallor, piloerection, bilateral semiclosed eyes, brown staining muzzle and swollen abdomen occurred for this female. At macroscopic observations findings observed were distended stomach, ileum, jejunum and caecum with gas and brown staining of muzzle. Microscopically squamous hyperplasia characterised by the presence of hyperkeratosis and associated with chronic inflammation of the submucosa of non-glandular stomach was noted. In light of the above macro and/or microscopic observations associated with hunched posture and/or swollen abdomen observed at clinical signs, the cause of the illness status or death for the two high dose females (nos. E0575025 and E0575029) were considered related to the administration of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Compared with control group, a slightly lower mean group body weight (statistically significant in the males only) was observed for males and females dosed at 500 mg/kg/day on Day 8 and Day 15 (up to -16%).

A treatment-related decrease in terminal body weight was observed in high dose animals of both sexes, statistically significant for high dose males (approximately -14%) and not statistically significant for high dose females (approximately -8%), when compared to the controls; this change correlated with decreased food consumption.

For details on body weight, please refer to Attachment 2 - DRF Summary table 2 under section "Attached background material."

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight reduction in food consumption was observed in the males dosed at 500 mg/kg/day on Days 8 and 15, when compared to controls (up to -19%). A transient reduction of food intake was seen in females dosed at 500 mg/kg/day on Day 8 (-47%), no longer observed on Day 15.

For details on food consumption, please refer to food consumption per animal/day data in appendix 4 of the study report under section "Attached background material."

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes in absolute and/or relative organ weights were observed in
high dose males, when compared to the controls. A statistically significant increase in absolute and/or relative mean adrenals weight (+34 % mean relative to brain weight) and decrease in heart (-15% mean relative to brain weight), kidneys (-8% mean relative to brain weight), liver (-14 % mean relative to brain weight), and thymus (-39% mean relative to brain weight) is considered attributed directly (adrenals and thymus) or indirectly (heart, kidneys and liver) to stress and/or as a consequence of decreased food consumption.

For details on organ weights, please refer to Attachment 2 - DRF Summary tables 3, 4 and 5 under section "Attached background material".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, one high dose male (1 out of 4) and the two surviving high dose females (2 out of 4) showed macroscopically thickened stomach. Microscopically, this corresponded to squamous hyperplasia of the forestomach. Any observations other than those listed above had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

For details on macroscopic findings, please refer to Attachment 2 - DRF Summary table 6 under section "Attached background material".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes, associated with the oral administration of Betaines, C12-14 (even numbered)-alkyldimethyl were present in the stomach (non-glandular region) of mid dose and high dose animals of both sexes. They consisted of squamous hyperplasia and were present with moderate to marked severity in all high dose males and surviving females.

A similar pathological picture was present with a lower severity (minimal to moderate) in all mid-dose males and females. The gastric lesion was characterized by convolutions of the epithelium with downward projections of rete peg-like structures associated with the presence of hyperkeratosis with chronic inflammation and/or oedema in the submucosa.

For details on microscopic findings, please refer to Attachment 2 - DRF Summary table 7 under section "Attached background material".

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In conclusion, Betaines, C12-14 (even numbered)-alkyldimethyl, when orally administered at dose levels of 30, 125 and 500 mg/kg/day for 2 consecutive weeks, resulted to be satisfactorily tolerated by Sprague Dawley rats at the low dose level (30 mg/kg/day). The highest dose level of 500 mg/kg/day induced some toxicity (mortality, clinical signs, reductions in body weight and food consumption, changes in terminal body weight and organ weights, gastric lesions found at post mortem macro and microscopic examination). At the intermediate dose level of 125 mg/kg/day, sporadic clinical signs, and gastric lesions with less severity compared to the high dose were observed. Therefore, it can be concluded that the intermediate and high dose, respectively 125 and 500 mg/kg/day, were not considered to be safe in a subsequent toxicity study of longer duration in Sprague Dawley rats.

Executive summary:

The toxicity of Betaines, C12-14 (even numbered)-alkyldimethyl, after daily oral administration (gavage) to Sprague Dawley SD rats at dose levels of 30, 125 and 500 mg/kg/day, was investigated over a period of 2 consecutive weeks. The study design, comprising 4 animals/sex/group, included a control group receiving the vehicle (softened water).

 

Excluding the early decedent at 125 mg/kg/day due to a misdosing, two females dosed at 500 mg/kg/day were found dead or sacrificed for humane reasons on Day 5. Based on the reported macro and/or microscopic observations, the preterminal death of the above animals was related to the administration of the test item.

 

Hunched posture, piloerection, swollen abdomen, decrease activity, rales, dyspnoea and salivation were the most relevant clinical signs observed in all males and/or surviving females of the high dose group (500 mg/kg/day) throughout the study. On occasion, hunched posture was observed in 3 females from mid-dose group (125 mg/kg/day).

 

Changes in body weight and food consumption were observed for high dose animals (500 mg/kg/day), with slightly lower mean group values compared to controls. At post mortem examination, treatment related decrease in terminal body weight was observed in high dose males and females, when compared to the controls, along with changes in absolute and/or relative (to brain weight) organ weight (increase in adrenal and decrease in heart, kidneys liver and thymus). The above findings could be attributed directly to stress and/or considered a consequence of decreased food consumption.

 

Microscopic examination, limited to the stomach of mid- and high dose groups, revealed treatment related squamous hyperplasia of the forestomach found in all animals with dose-related severity. This finding was consistent with the reported gross observations (thickened stomach in one male and two females of high dose groups).

 

In conclusion, Betaines, C12-14 (even numbered)-alkyldimethyl, when orally administered at dose levels of 30, 125 and 500 mg/kg/day for 2 consecutive weeks, resulted to be satisfactorily tolerated by Sprague Dawley rats at the low dose level (30 mg/kg/day). The highest dose level of 500 mg/kg/day induced some toxicity (mortality, clinical signs, reductions in body weight and food consumption, changes in terminal body weight and organ weights, gastric lesions found at post mortem macroscopic and microscopic examinations). At the intermediate dose level of 125 mg/kg/day, sporadic clinical signs, and gastric lesions with less severity compared to the high dose were observed. Therefore, it can be concluded that the intermediate and high dose, 125 and 500 mg/kg/day respectively, were not considered to be safe in a subsequent toxicity study of longer duration in Sprague Dawley rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Betaines, C12-14 (even numbered)-alkyldimethyl

EC Number:

931-700-2

Cas Number:

66455-29-6

Molecular formula:

C16H33NO2 C18H37NO2

IUPAC Name:

Betaines, C12-14 (even numbered)-alkyldimethyl

Test material form:

liquid

Details on test material:

Batch: BLBX030618
Manufacture date: 05.01.2020
Expiry Date: 04.01.2022
Appearance: Clear Liquid
Free Amine (%): 0.18
Active Matter (%): 29.92
pH: 7.2
Sodium Chloride (%): 7.11
Sodium Monochloride (ppm): 5
Colour (Hazen): 12
C-Chain analysis of batch BLBX030618 (Subs ID Report RT0158S) show that material fits the REACH substance Identity profile.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Source: Innospec Limited
Identity: Betaines, C12-14 (even numbered)-alkyldimethyl
Label name: EMPIGEN® BB/FL
EC number: 931-700-2
Batch no: BLBX030618
Retest date: 04 January 2022
Appearance: Clear liquid
Purity: 29.92%
Correction factor 3.34

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition of test material: Room temperature, protected from light and moisture
Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: preparation was made at weekly intervals at concentrations of 1, 3 and 10 mg/mL and stored at room temperature for a maximum of 8 days, based on results from ERBC Study no. A4171 (28 hour and an 8 day stability at room temperature were verified in the range from 1 to 100 mg/mL).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Treatment of test material prior to testing: N/A

OTHER SPECIFICS
C-Chain analysis of batch BLBX030618 (Subs ID Report RT0158S) show that material fits the REACH substance Identity profile.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD 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 B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 27-29 days old
- Weight at study initiation: 90-100 g for males, 82-91 g for females
- Housing: The animals were housed in a limited access rodent facility. The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water (e.g. ad libitum): ad libitum to each cage via water bottles
- Acclimation period: 19 days

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. Records of analysis of water, diet and bedding material are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

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

On the day of allocation (6 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 4 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group will be evenly distributed across the battery to minimise possible environmental effects.

IN-LIFE DATES:
Start of experimental phase (ERBC) - Allocation of animals to the study: 16 February 2021
End of experimental phase (ERBC) - Last day of necropsy: 23 June 2021

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight/day. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Vehicle:

water

Details on oral exposure:

All animals were dosed orally by gavage, once a day, 7 days a week, for a minimum of 13 consecutive weeks, followed by a recovery period of at least 4 weeks for 10 males and 10 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy.

PREPARATION OF DOSING SOLUTIONS:
Dosing concentrations were prepared weekly at concentrations of 1, 3 and 10 mg/mL
and stored at room temperature for a maximum of 8 days, based on results from ERBC Study
no. A4171. Concentrations were calculated and expressed in terms of test item corrected
for purity (correction factor 3.34).

VEHICLE
- Vehicle: softened water (by reverse osmosis)
- Concentration in vehicle: 1, 3 and 10 mg/mL for low-, mid- and high-dose group, respectively.

Analytical verification of doses or concentrations:

yes

Remarks:

Samples of the preparations prepared on Weeks 1 and 13 were analysed to check the concentration.

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4171 in the range from 1 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 90-110%; precision CV < 5%). In ERBC Study no. A4171, a 28 hour and an 8 day stability at room temperature were verified in the range from 1 to 100 mg/mL. According to ERBC SOPs, solutions are considered to be stable if concentration, after the defined period of storage, is still acceptable (90-110%). The proposed formulation procedure for the test item was checked in the range from 1 to 100 mg/mL by chemical analysis (concentration) in ERBC Study no. A4171 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in the ERBC SOPs for concentration (90-110%). Samples of the preparations prepared onWeeks 1 and 13 were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for concentration of solutions (90-110%). The validated software used for this activity was Analyst 1.6.2.

Duration of treatment / exposure:

Animals were dosed for a minimum of 13 consecutive weeks, followed by a recovery period of at least 4 weeks for 10 males and 10 females from groups 1 and 4.

Frequency of treatment:

Once a day, 7 days a week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats. Control and high dose groups included 10 additional animals per sex to be sacrificed after 4 weeks of recovery.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for the study were selected based on information from preliminary non-GLP study ERBC No. E0575, see DRF endpoint record. In this 2 Week Preliminary Oral Toxicity Study, Betaines, C12-14 (even numbered)-alkyldimethyl was administered at dose levels of 30, 125 and 500 mg/kg bw/day. The highest dose level of 500 mg/kg bw/day induced some toxicity (mortality, clinical signs, reductions in body weight and variations in food consumption, changes in terminal bodyweight and absolute/relative organ weight, findings at post mortem examination involving the gastric tract. On occasion, clinical signs were observed at the mid-dose level (125 mg/kg bw/day). Therefore, the high dose of 500 mg/kg bw/day, was not considered to be safe in a subsequent longer duration toxicity study. A high dose of 100 mg/kg bw/day was supposed to be reasonably safe, although some toxicity was expected. Dosages of 10 and 30 mg/kg bw/day were selected for low and mid-dose levels.

- Fasting period before blood sampling for clinical biochemistry: yes; except for blood samples taken on Day 30 from the second half of the satellite animals/ group, that were not fasting. However, since food was only available to these animals for circa 1 hour before withdrawal, this event was not considered to have affected the haematological, biochemical and thyroid hormone results.

- Post-exposure recovery period in satellite groups: 4 weeks

- Dose range finding studies: 14-day preliminary non-GLP study ERBC No. E0575, see DRF endpoint record

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality: all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day.
Clinical signs: Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day (0.5-1h post-dose), the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once per week during the study from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation and record of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil response, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.

BODY WEIGHT: Yes
Time schedule for examinations: On the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
Food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals were examined prior to the commencement of treatment. The eyes of all animals from high-dose and control groups were re-examined during week 13 of treatment.

HAEMATOLOGY: Yes
Time schedule for collection of blood: During the last week of treatment
Anaesthetic used for blood collection: Yes (isoflurane)
Animals fasted: Yes – with the exception of blood samples taken on Day 30 of recovery, were not collected from fasting animals. However, since food was only available to these animals for circa 1 hour before withdrawal, this event was not considered to have affected the results.
How many animals: all animals
Parameters checked: Haematocrit (HCT), Haemoglobin (HGB), Red blood cell count (RBC), Reticulocyte count (relative and absolute), Mean red blood cell volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), White blood cell count (WBC), Differential leucocyte count (relative and absolute) - Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells. Platelets. These parameters were analysed by Siemens Advia 120.
Coagulation: Prothrombin time. This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: During the last week of treatment
Animals fasted: Yes - with the exception of blood samples taken on Day 30 of recovery, were not collected from fasting animals. However, since food was only available to these animals for circa 1 hour before withdrawal, this event was not considered to have affected the results.
How many animals: all animals
Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyltransferase (GGT), Blood urea nitrogen (BUN), Urea, Creatinine (CREA), Bile acids (ACBIL), Glucose (GLU), Triglycerides (Trig), Inorganic phosphorus (IP), Total bilirubin (TBIL), Total cholesterol (CHOL), HDL, LDL, Total protein (PROT), Albumin (ALB), Globulin (GLO), A/G Ratio (A/G), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl).
These parameters were analysed by Siemens Advia 1800.

PLASMA/SERUM HORMONES/LIPIDS: Yes
Time of blood sample collection: during the last week of treatment
Animals fasted: Yes
How many animals: all main phase animals
Parameters checked: Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH).
Thyroid hormone determination: RV-T3/R/S-BKM/RIA-002 for T3, RV-T4/R/S-BKM/RIA-002 for T4 and RV-TSH/R/S-IZO/RIA-002 for TSH

URINALYSIS: Yes
Time schedule for collection of urine: during the last week of treatment
Metabolism cages used for collection of urine: Not specified
How many animals: all animals
Animals fasted: Yes, overnight
Parameters checked: Volume, Appearance, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood. These parameters were analysed byMenarini AutionMax AX 4280/Aution Sticks 10EA and ATAGO Refractometer (for Specific gravity), according to internal procedures.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors and other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: Several functional tests, such as hind limb landing foot splay, sensory reactivity to stimuli, including grip strength and motor activity, were performed once during Week 12 of treatment and at the end of the recovery period, for neurotoxicity assessment.
Dose groups that were examined: all animals

IMMUNOLOGY: No

OTHER:
OESTRUS CYCLE EVALUATION: Vaginal smears were taken from all females during the last 2 weeks of dosing and at the end of the treatment and recovery periods, just prior to necropsy.

SPERM ANALYSIS: During the necropsy procedure, one cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility and morphology. The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment. The animals of the mid- and low dose groups were dosed until the evaluation of the control and high dose animals was performed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Terminal studies
Animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals, including those found dead, were subjected to necropsy, supervised by a pathologist. The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals completing the scheduled test period, the organs indicated in Attachment 1, Annex 1 under section "Attached background material” were dissected free of fat and weighed. The ratios of organ weight to body weight was calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in Attachment 1, Annex 1 under section "Attached background material” were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGY: Yes

Histopathological examination
The tissues required for histopathological examination are listed in Attachment 1, Annex 1 under section "Attached background material”. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment. Sections from the vagina, uterus and ovaries from the control and high dose females killed at the end of the 13 weeks of treatment were examined for evaluation of oestrus cycle.

In the first instance the examination was as detailed below:
i) Tissues specified in attachment 1 from all animals in the control and high dose groups, dying during the treatment period or killed at the end of the 13 weeks of treatment.
ii) Tissues specified in attachment 1 from all animals killed or dying during the study.
iii) All abnormalities in all main phase groups.

On the basis of treatment-related changes observed in the mucosa of non-glandular region of the stomach in males and females receiving Betaines, C12-14 (even numbered)- alkyldimethyl at 100 mg/kg bw/day the histopathological evaluation was extended to the remaining main and recovery phase animals.

Optional endpoint(s):

Optional endpoints: Yes

Urinalysis: During the last week of treatment, individual overnight urine samples were collected from all male and female animals from each main phase group under conditions of food and water deprivation. Appearance, Volume (manually recorded), Specific gravity, pH, Protein, Parameters assessed: Glucose, Ketones, Bilirubin, Urobilinogen, Blood. These parameters were analysed by Menarini AutionMax AX 4280/Aution Sticks 10EA and ATAGO Refractometer (for Specific gravity), according to internal procedures.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors and other abnormal components.

Spermanalysis: During the necropsy procedure, one cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility and morphology. The animals of the mid- and low dose groups were dosed until the evaluation of the control and high dose animals was performed.

Oestrus cycle: Vaginal smears were taken from all females during the last 2 weeks of dosing to evaluate effects on Oestrus cycle and at the end of the treatment and recovery periods, just prior to necropsy.

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 inhomogeneous a Modified t test (Cochran and Cox) was applied.

The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were observed during the study. Salivation was occasionally observed in one male animal treated at 30mg/kg/day on Days 72 and 73 of the dosing period; rales were observed in one male animal treated at 100mg/kg/day starting from Day 32 up to Day 48 of the dosing period. Missing teeth was occasionally observed in one female of the control group while another female of the same group showed a palpable mass on Days 91 and 92. Sporadic dyspnoea, moderate pale and rales were observed in one female of the high group (100 mg/kg/day).

During the recovery period, slight brown staining was observed on both eyelids of one male of the high dose group. All the above clinical signs, due to their sporadic occurrence, were not considered to be adverse.

Observation of animals at removal from the cage and in an open arena did not reveal treatment-related changes. At the weekly clinical examination, which included an evaluation of neurotoxicity, few differences were noted among groups. Statistically significant decreases or increases in rearing were recorded in males at 30 and 100mg/kg bw/day and in females treated at 10, 30 and 100mg/kg bw/day at different timepoints of the dosing period. A statistically significant decrease in defecation was observed in females treated at 30 and 100mg/kg bw/day on Day 88 of the dosing period. During the recovery period, decrease in rearing was recorded in males of the high group on Days 3 and 10 (up to -20%) and in females on Day 11 (-13%). These findings were mostly not dose-related and were not consistent, therefore not considered to be treatment-related.

For details on clinical signs, please refer to Attachment 2 - Tables 1 and 2 under section "Attached background material".

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two high dose males, nos. A4172102 and A4172108, were found dead on Day 15 and on Day 72 of the study, respectively. No clinical signs were observed in these animals. At post mortem examination male no. A4172102 had dark red fluid in the thoracic cavity and the cause of death was consistent with misdosing. Male no. A4172108 had swollen liver with multiple dark red, up to 3x2mm areas. Microscopically, mild congestion was present in the liver and lungs. These observations did not suggest any cause or factors contributory to death for this male.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

When compared to control animals, no changes were noted in mean body weights in both genders, during dosing and recovery periods.

For details on body weight and body weight gains, please refer to Attachment 2 - Figure 2 and Tables 6 - 7 under section "Attached background material."

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes were observed in food consumption in male and female animals, during the dosing and recovery periods.

For details on food consumption, please refer to Attachment 2 - Table 8 under section "Attached background material."

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Before the start of treatment, animals showing no ocular abnormality at the ophthalmoscopic examination were selected for the study. Both eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment (Study Day 91). No ocular findings were detected.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were recorded. The statistically significant differences between control and treated females (erythrocytes, red distribution width, haemoglobin, haematocrit and basophils) were recorded in animals dosed at 10 and/or 30 mg/kg/day, therefore considered to be incidental.
For details on haematology, please refer to Attachment 2 - Table 10 under section "Attached background material".

Coagulation:
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
For details on coagulation, please refer to Attachment 2 - Table 11 under section "Attached background material".

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were recorded.

Males dosed at 30 and 10 mg/kg bw/day showed an increase of LDL (21% and 27%, respectively) and a decrease of triglycerides (37% and 41%, respectively). Changes were within the range of expected biological variation and no other related findings were recorded (e.g. histopatological hepatic changes), therefore they were considered to be unrelated to treatment. Protein was statistically significantly higher than controls in females dosed at 100 mg/kg bw/day (4%). Values were within the range of expected biological variation and no other related finding was recorded, therefore this change was considered to be unrelated to treatment. The other statistically significant differences recorded between control and treated animals (aspartate aminotransferase, creatinine and potassium in males, alanine aminotransferase, calcium and potassium in females) were recorded in animals dosed at 10 and/or 30 mg/kg bw/day, therefore considered to be incidental.

Following the recovery phase no changes were recorded, confirming complete reversibility.The statistically significant differences recorded between control and treated males (bilirubin, blood urea nitrogen, chloride and albumin) were not recorded at the dosing phase, therefore they were considered to be incidental.

For details on clinical biochemistry, please refer to Attachment 2 - Table 12 under section "Attached background material".

Endocrine findings:

no effects observed

Description (incidence and severity):

The determination of T3, T4 and TSH was performed in the main phase animals. No differences between control and treated animals were recorded. For details on thyroid hormones, please refer to Attachment 2 - Table 14 under section "Attached background material".

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were recorded. For details urinalysis, please refer to Attachment 2 - Table 13 under section "Attached background material".

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Several functional tests, such as hind limb landing foot splay, sensory reactivity to stimuli, including grip strength and motor activity, were performed during Week 12 of treatment and at the end of the recovery period, for neurotoxicity assessment. Only few differences among groups were noted in landing foot splay, motor activity and sensory reactivity to stimuli results. A statistically significant decrease in landing foot splay was recorded in males treated at 30 and 100mg/kg bw/day on Week 12 of the dosing period. A statistically significant decrease in the first measurement of grip strength was recorded in males treated at 100mg/kg bw/day on Week 12 of the treatment period. Since these signs occurred only occasionally in individual animals and due to the direction of some of these changes (decrease in landing foot splay), they were not considered to be treatment-related. Motor activity measurements performed at the end of the dosing period did not show any differences between dosed animals and controls.
Recovery phase: A statistically significant increase in motor activity was recorded in males and females of the high group at the end of the recovery period. This change, not observed during treatment period, was considered to be incidental.

For details on functional behaviour, please refer to Attachment 2 - Tables 3 - 5 under section "Attached background material

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in terminal body weights in treated males and females, when compared to their controls. Slightly increased absolute (+9%) and relative (+10%) kidney weights and increased absolute (+8%) and relative (+9%) liver weights were noted in Group 4 females when compared with controls. These organ weight changes were considered to have no toxicological relevance considering their magnitude and the absence of corresponding microscopic observations.

Recovery sacrifice: At the end of the recovery period, there were no treatment-related change in the terminal body weight. In addition, there was complete reversibility of changes observed at the end of the treatment period in the liver and kidney weights. Any organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice: At the end of the treatment period, treatment-related macroscopic findings were present in the stomach of males receiving Betaines, C12-14 (even numbered)-alkyldimethyl at 100 mg/kg bw/day. The gastric macroscopic changes consisted of thickened non glandular mucosa (forestomach) and/or occasional presence of multiple dark area/s in the mucosa of glandular region. No macroscopic findings were noted in the stomach at doses 10 or 30 mg/k/day. Any other macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

Recovery sacrifice: At the end of the recovery period, there were no treatment-related macroscopic findings in males and females receiving Betaines, C12-14 (even numbered)-alkyldimethyl. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

For details on macroscopic findings, please refer to Attachment 2 - Table 17 under section "Attached background material".

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice: At the end of the treatment period, treatment-related changes were only noted in males and females receiving Betaines, C12-14 (even numbered)-alkyldimethyl at 100mg/kg bw/day. The pathological changes were present in the forestomach and consisted in generalized epithelial cell hyperplasia of the stratified squamous layer associated with hyperkeratosis and/or focal mucosal erosion with submucosal infiltration of mononuclear cells, proliferation of fibroblasts and oedema (chronic inflammation). Males and females receiving Betaines, C12-14 (even numbered)-alkyldimethyl at 10 (Group 2) or 30 (Group 3) mg/kg bw/day did not show treatment-related changes in the stomach (forestomach). There were no treatment related microscopic observations in the testes examined with PAS special stain. There were no treatment-related effects on physiology of the estrous cycle (estrus, metestrus, dioestrus and proestrus) in control and high dose females.
Any other microscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

Recovery sacrifice: After 4 weeks of recovery period, there were no microscopic observations in the stomach of males and females receiving Betaines, C12-14 (even numbered)-alkyldimethyl at 100 mg/kg bw/day indicating full reversibility of the changes observed at the end of the treatment period.

For details on microscopic findings, please refer to Attachment 2 - Table 18 under section "Attached background material".

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Oestrus cycle:
Vaginal smears were taken from all females during the last 2 weeks of dosing in order to evaluate test item effects on Oestrus cycle. No treatment-related anomalies were observed in the oestrus cycle of the treated females during the last 2 weeks of the treatment period and at the end of recovery phase.

For details on Oestrus cycle, please refer to Appendix 12 in the full report provided in attachment 3 under section "Attached background material".

Sperm analysis:
During the necropsy procedure, one cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility and morphology.

No differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end of treatment.

For details on sperm analysis, please refer to Attachment 2 - Table 19 under section "Attached background material".

Details on results:

For further details on results, please refer to attachment 2, under section 'attached background material'.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: highest dose tested

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Slight signs of treatment-related effects were observed at postmortem observations in animals dosed at 100 mg/kg bw/day. These changes (very slight increases in liver and kidneys weight) were not considered adverse due to their magnitude and in the absence of corresponding microscopic observations. At macroscopic and microscopic examinations, changes were observed at the end of treatment period in the forestomach of animals dosed at 100mg/kg bw/day. These changes were no longer present at the end of recovery period. Changes in the stomach could be ascribed to a local irritant effect of the test item. They were not sufficiently severe to become adverse, fully reversible and not relevant for humans due to their location. No treatment-related changes were observed in animals dosed at 10 and 30 mg/kg bw/day. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 100mg/kg bw/day.

Executive summary:

The toxicity of Betaines, C12-14 (even numbered)-alkyldimethyl was investigated in rats after daily oral administration for 13 consecutive weeks and recovery from any treatment related effects during a recovery period of 4 weeks.

Two high dose males were found dead on Day 15 and on Day 72 of the study, respectively. No clinical signs were observed in these animals. The cause of death was consistent with misdosing for one male, while macroscopic and microscopic observations did not suggest any cause or factors contributory to death for the other male. No treatment-related clinical signs or changes in body weight, body weight gain and food consumption were observed during the study. No adverse changes were observed in functional or motor activity measurements. No treatment-related ocular findings or effects on estrous cycle or sperm analysis were observed. No treatment-related changes were recorded in haematological, coagulation, clinical chemistry, thyroid hormone and urinalysis parameters. No changes of toxicological significance were observed on terminal body weight, absolute and relative organ weights in the main and recovery phase animals.

Treatment-related macroscopic findings were present in the stomach of males receiving Betaines, C12-14 (even numbered)-alkyldimethyl at 100 mg/kg bw/day. The gastric macroscopic changes consisted of thickened non-glandular mucosa (forestomach) and/or, occasionally, presence of multiple dark area/s in the glandular region, which were fully reversible within the recovery period. Treatment-related microscopic changes that were fully reversible during the recovery period were noted in both sexes receiving 100mg/kg bw/day. The pathological changes were present in the forestomach and consisted of generalized epithelial cell hyperplasia of the stratified squamous layer associated with hyperkeratosis and/or focal mucosal erosion with submucosal infiltration of mononuclear cells, proliferation of fibroblasts and oedema (chronic inflammation). Males and females receiving 10 or 30 mg/kg bw/day did not show treatment-related changes in the stomach (forestomach).

In conclusion, some changes were observed at postmortem observations in animals dosed at 100 mg/kg bw/day. These changes (very slight increases in liver and kidneys weight) were not considered adverse due to their magnitude and in the absence of corresponding microscopic observations. Changes in the stomach could be ascribed to a local irritant effect of the test item. They were not sufficiently severe to become adverse, fully reversible and not relevant for humans due to their location. No treatment-related changes were observed in animals dosed at 10 and 30 mg/kg bw/day. Therefore, it was concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 100mg/kg bw/day.