Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

OECD 408 (Repeated Dose 90-Day Oral): NOAELrat = 100 mg/kg bw/day a.i.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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:
other: GLP guideline study. Reliability changed from "1" to "2" according to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010).
Justification for type of information:
This study was conducted on a mixture of lauryl betaine and myristyl betaine which are constituents of the registered (target) substance. See section 13 for the full read-across justification.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Fuellinsdorf, Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: males: 296 - 330 g; females: 180 - 212 g
- Fasting period before study: no data
- Housing: individually; during the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if: a) The daily vaginal smear was sperm positive, or b) A copulation plug was observed.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no. 65/07 rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water (e.g. ad libitum): Community tap-water from Fuellinsdorf, ad libitum in water bottles.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose levels were in terms of the a.i. in the aqueous solution. A correction factor of 3.765 was used. The dose formulations were prepared weekly, using the test item as supplied by the Sponsor. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Concentration in vehicle: 5, 15, 30 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test substance in milli-Q-water was demonstrated. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
Duration of treatment / exposure:
Males: Minimum 4 weeks; females: Approximately 7 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day (active ingredient)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, RCC Study Number B62763.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, during acclimatization and up to day of necropsy; viability/mortality twice daily
- Cage side observations included: clinical signs; females additionally for signs of difficult or prolonged parturition and behavioural abnormalities in nesting and nursing


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and daily thereafter. Observations included: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: Daily from treatment start to day of necropsy.


FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum. No food consumption was recorded during the pairing period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 males from each group, 5 lactating females from each group
- Parameters examined: Complete Blood Cell Count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total), Differential leukocyte count, Platelet count, Coagulation, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 males from each group, 5 lactating females from each group
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 P generation males shortly before the scheduled sacrifice and 5 P generation females on day 3 or 4 post partum, following the daily dose administration
- Dose groups that were examined: each group
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (were made at any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®). All P generation animals were exsanguinated. Dead pups were examined macroscopically. All parent animals and pups were examined macroscopically for any structural changes. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.
HISTOPATHOLOGY: Yes, Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative) and ovaries from all parental animals. In addition, from the five males and females per group selected for organ weights, the following tissues were preserved: Gross lesions, Brain, Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroids, Trachea and lungs (preserved by inflation with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic), Bone marrow.

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS-hematoxylin.

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Due to possible test item-related findings noted in the high dose group, the kidneys, urinary bladder, stomach (forestomach and glandular stomach), cecum, colon, rectum, trachea, lungs, bone marrow and adrenal glands were examined also in the animals in groups 2 and 3. Histological examination of ovaries was carried out on females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.
Other examinations:
Organ Weights: The testes and epididymides of all parental males were weighed as pairs. In addition, from 5 males and females selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, Spleen.
Statistics:
Fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices were calculated from online recorded data. For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

The following statistical methods were used for food consumption, body weight, macroscopical findings, organ weights and reproduction data:
Means and standard deviations, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution. Fisher's exact-test was applied to the macroscopical findings.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until the scheduled necropsy.

300 mg/kg bw/d:
Incidences of ruffled fur (from second week onwards, slightly ruffled in 4 males and all females, and one male had ruffled fur over the whole body), diarrhea (3 males and 3 females in the second week), sedation (all animals in the second and third week), salivation and pushing their head through the bedding occurred from day 5/6 of the pre-pairing period up to the end of the treatment, either periodically or continuously in the males and females, as well as salivation in some individuals. Four males were in a bad condition for up to 5 days during the pre-pairing period. The feces from all males and 4 females were noted to be round and soft in comparison to the control group. One female had loose feces. Several puddles of urine were noted for all males and females. In addition, mean body temperature was statistically significantly reduced in both males and females. All these findings were considered to be test item-related.

150 mg/kg bw/d:
All males and females pushed their head through the bedding after application from day 7/8 of the pre-pairing period onwards. Male no. 27 salivated periodically during the prepairing and pairing periods. All males salivated during the last 3 or 4 days of the after pairing period. Female no. 65 had salivation during the last part of the gestation period. All of the above were considered to be test item-related, with the exception of pushing the head through the bedding. This was considered to be a sign of discomfort following application rather than a toxic effect of the test item.

Control group and 50 mg/kg bw/d:
No clinical signs or symptoms were noted for any animal in any group during the study.

BODY WEIGHT AND WEIGHT GAIN
Males (Pre-pairing, Pairing and After Pairing Periods):
300 mg/kg bw/d:
Body weight gain was statistically significantly reduced from day 2 until the end of the pre-pairing period and absolute body weight from day 3 onwards. Over the whole of the pre-pairing period, males increased in weight by 1.6% compared to 12.9% in the control group. During the pairing and after pairing periods, body weight gain was not considered to have been affected by treatment with the test item but absolute body weight remained statistically significantly reduced until the end of the study period.

150 mg/kg bw/d:
Mean body weight gain was slightly reduced over the pre-pairing period (+9.8% compared to 12.9%). Thereafter, it was similar to that of the control group. Absolute body weight became reduced in the pre-pairing period and remained so for the rest of the study compared to the control group but was at no time statistically significantly reduced.

50 mg/kg bw/d:
Mean body weight and body weight gain were similar to that of the control group.

Females (Pre-pairing, Pairing, Gestation and Lactation Periods):
300 mg/kg bw/d:
Mean body weight gain was statistically significantly reduced from day 2 until day 13 and absolute body weight from day 3 until day 11 of the pre-pairing period (+6.6% compared to +9.7% in the control group over days 1 - 14). Thereafter, it was similar to that of the control group, with the exception of days 14 - 21 of the gestation period (+13.1% compared to +29.5% in the control group). Mean absolute body weight was statistically significantly reduced from day 18 - 21 of the gestation period.

150 and 50 mg/kg bw/d:
Mean body weight and body weight gain were not considered to have been affected by treatment with the test item.

FOOD CONSUMPTION
Males (Pre-pairing and After Pairing Periods):
300 mg/kg bw/d:
Mean food consumption was statistically significantly reduced during the first week of the pre-pairing period (-44.5% compared to the control group over days 1 - 8). This was considered to be related to treatment with the test item. Food consumption recovered thereafter.

150 mg/kg bw/d:
Mean food consumption was reduced over days 1 - 8 (-11.7% compared to the control group). Although this reduction was not statistically significant, it was considered to be a test item-related effect. It remained reduced thereafter but there was no dose-dependent reduction.

50 mg/kg bw/d:
Food consumption was similar to that of the control group during the whole of the study.

Females (Pre-pairing, Gestation and Lactation Periods):
300 mg/kg bw/:
Mean food consumption was statistically significantly reduced over days 1 - 8 of the pre-pairing period (-37.5% compared to the control group) but recovered in the second week. In the gestation period, food consumption was again statistically significantly reduced over days 14 - 21 (-16.4% compared to the control group) and over the whole of the lactation period (-23.8% compared to the control group). This was considered to be related to treatment with the test item.

150 mg/kg bw/d:
Mean food consumption was reduced, but not statistically significantly over days 1 - 8 of the pre-pairing period (-10.7% compared to the control group). Thereafter it recovered and was similar to that of the control group.

50 mg/kg bw/d:
Mean food consumption was similar to the control group for the whole of the study.


HAEMATOLOGY
Males:
No test item-related findings were noted. All statistically significant differences (number of hemoglobin and neutrophils, and activated partial thromboplastin time in the 300 mg/kg group and the mean corpuscular hemoglobin concentration in the 150 and 300 mg/kg groups) were all within the range of the historical control data.

Females:
No test item-related findings were noted. No statistically significant differences were noted between the control group and the groups receiving the test item.

CLINICAL CHEMISTRY
Males:
The measurement of urea was statistically significantly increased in the 150 and 300 mg/kg groups. This was outside the range of the historical control data and was considered to be test item-related. The statistically significantly reduced level of glucose in the 300 mg/kg group and increase of triglycerides in the 150 mg/kg group were considered to be incidental.

Females:
No test item-related findings were noted. No statistically significant differences were noted between the control group and the groups receiving the test item.


NEUROBEHAVIOUR
300 mg/kg bw/d:
3 females had a decreased number of rearings and one male had an increased number of rearings. This was considered to be incidental. The reduction in landing foot distance in the females at this dose level was not statistically significant and was considered to be incidental due to the lack of dose-dependency.

150 and 50 mg/kg bw/d:
No findings were noted which were considered to be test item-related.

Locomotor activity was assessed quantitatively in terms of low beam counts in an activity monitor.
300 mg/kg bw/d:
The total activity was reduced in males and females, and although it was not statistically significantly reduced, it was considered to be test item-related.

150 and 50 mg/kg bw/d:
No test item-related effects were noted.

ORGAN WEIGHTS
300 mg/kg bw/d:
The organ/body weight ratios of the kidneys, liver and adrenals were slightly increased and the thymus slightly decreased in both males and females.

150 mg/kg bw/d:
The organ/body weight ratio of the liver was slightly increased in the males and females.

50 mg/kg bw/d:
No test item-related findings were noted.

GROSS PATHOLOGY
300 mg/kg bw/d:
A thickened stomach was noted in 4 males. One male had enlarged adrenal glands. A thickened stomach was noted in 4 females and crateriform retractions in 2 females. The lungs of one female did not collapse at necropsy. This was considered likely to be due to aspiration of the test item.

150 mg/kg bw/d:
A thickened stomach was noted in one male, retractions of the stomach were noted in one female.

50 mg/kg bw/d:
Foci were found on the stomach of one female.

All these macroscopical findings correlated with microscopical findings and were considered to be test item-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Under the conditions of this study, test item-related changes in the kidneys, urinary bladder, stomach, adrenal glands and bone marrow were observed. In the kidneys, urothelial and collecting duct hyperplasia occurred in high dose group animals along with granular casts and an increased incidence and severity of tubular basophilia and mononuclear cell infiltrates. Minimal granular casts were also found in one mid dose male. Urothelial hyperplasia in the urinary bladder was recorded in one male of the high dose group and in females of the mid and high dose groups.

In the stomach, various degrees of ulceration/erosion, squamous hyperplasia, hyperkeratosis, parakeratosis, pustules, submucosal inflammation and edema were recorded in the forestomach of animals of all dose groups. Mucosal necrosis as well as submucosal edema and inflammation were found randomly distributed in the glandular stomach of mid and high dose group animals.

The histopathologic changes in the kidney and the urinary bladder observed in the mid and high dose groups were consistent with possible irritant effect of the test article and/or its metabolite excreted in the urine. The lesions observed in the forestomach in all dose groups and in the glandular stomach of some individuals in the mid and high dose groups represented a localized stomach reaction to a repeatedly gavaged irritant test material. These changes noted in the kidneys, bladder and stomach were considered adverse.

In addition, there was a slight increase in the incidence and severity of extramedullary hematopoiesis in the adrenal glands (cortices) of females of the high dose group. This was interpreted to be secondary to the inflammatory response in the stomach and possible loss of small quantities of blood through the gastric erosions/ulcers. The slightly increased granulopoiesis in the bone marrow observed in occasional females of the low dose group, and males and females of the mid and high dose groups were interpreted to be secondary to the inflammatory response in the stomach. The effects noted in the adrenals and bone marrow were thus considered adaptive and therefore, not adverse.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs (salivation), clinical chemistry (increased urea), histopathology (kidney, bladder)
Dose descriptor:
NOAEL
Remarks:
reproduction/developmental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Dose descriptor:
LOAEL
Remarks:
reproduction/developmental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: pup weight, litter size, post-implantation loss, postnatal loss
Critical effects observed:
not specified

Conclusion:

The NOAEL for systemic toxicity was determined to be 50 mg/kg bw/d, as the adverse effects observed here were typical localized reactions to the repeated gavage of an irritant test material and don't have to be considered systemic. Therefore, the LOAEL for systemic toxicity was 150 mg/kg bw/d under the conditions of this study.

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
Reason / purpose for cross-reference:
reference to other study
Remarks:
Reference to dose range finding study
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
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: 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
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.
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 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
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.
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'.
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
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
Critical effects observed:
no
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available data comprises adequate and reliable (Klimisch score 2, either based on actual quality or reduced from score 1 due to read across) studies from reference substances with similar structure and intrinsic properties.
The selected study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII-IX, 8.6 in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Oral:

In a 90 day GLP study according to OECD test guideline 408 conducted with the registered substance, sprague-Dawley rats (10/sex/group) were administered Betaines, C12-14 (even numbered)-alkyldimethyl (EC 931-700-2) by oral gavage, daily for 13 weeks, at dose levels: 0, 10, 30 or 100 mg/kg bw/day followed by a recovery period of 4 weeks. 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. High-dose animals displayed thickened forestomach mucosa, considered a consequence of local toxicity (not systemic), which was reversible in a 28-day recovery period. All other observed changes in the high-dose group (including very slight increases in liver and kidney weights in the absence of corresponding macro- or microscopic changes) were deemed non-adverse. 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.

There is data available from a 90-day Repeated Dose Toxicity Study via the oral route with the structurally closely related substance CAS 68424-94-2 according to OECD guideline 408 in rats (Pittermann, 1993) and from two Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Tests according to OECD guideline 422, the first with a reaction mass containing both CAS 683-10-3 and CAS 2601-33-4 (Whitlow et al., 2009) with respective range-finder (Whitlow and Flade, 2008), and the second with lauryl betaine (CAS 683-10-3), the principle component of the registered substance (JECDB, 2005).


In the subchronic 90-day study (Pittermann, 1993) doses of 125, 250 and 500 mg/kg bw/day of a test material containing 29 - 33% CAS 68424-94-2 were administered to rats daily by oral gavage. In the course of the study no substance-related mortality occurred, and no substance-related clinical signs were observed in the exposed groups. The only treatment-related signs were a non-adverse dose-related increase in water consumption of the males and the high dose females, and a slight reduction of mean body weights of the high dose males from week 10 until the end of the study. All other parameters investigated showed no or only slight variations compared to the control group. Therefore, the mid-dose of 250 mg/kg bw/day was determined as NOEL, whereas the high-dose of 500 mg/kg bw/day was determined as LOEL. Considering the absence of other systemic effects like changes in clinical chemistry parameters, the LOEL can be regarded, at the same time, as NOAEL, as well. Converted to active ingredient this dose corresponds to 145 mg/kg bw/day, based on the assumption of 29% active ingredient in the test material. There were no adverse effects on reproductive organs reported in this study.


The first combined repeated dose/reproductive toxicity study was performed with a test substance consisting of 19.84% CAS 683-10-3 and 6.72% CAS 2601-33-4 (Whitlow et al., 2009). The test substance was applied to rats for 49 days by oral gavage. No systemic effects were observed in the low dose group, whereas signs of systemic toxicity like salivation, increased urea and histopathological findings in kidney and bladder were observed at the mid dose of 150 mg/kg bw/day. Therefore, the low dose of 50 mg/kg bw/day was determined as NOAEL, the corresponding LOAEL for systemic toxicity was 150 mg/kg bw/day. The corresponding range-finder (Whitlow and Flade, 2008) revealed a NOAEL of 100 mg/kg bw/day, the LOAEL of 300 mg/kg bw/day was characterised by signs of sedation, significantly reduced food consumption and body weight gain, very likely due to the fact that animals were sedated for most of the pre-pairing period. At 1000 mg/kg bw/day all animals died within 24 hours after the first treatment.


The second OECD 422 study was conducted on lauryl betaine (CAS 683-10-3). The test substance was administered daily by oral gavage for at least 42 days. Tested doses were 10, 60 and 300 mg/kg bw/day. There were no treatment-related effects on growth, food consumption, haematology, urinalysis (males only), neurobehaviour or organ weights. The critical effects were hyperplasia and necrosis of the kidney, along with bladder hyperplasia observed upon histological assessment of animals from the mid- and high-dose groups. Additional microscopic changes were noted in the forestomach of both sexes at the highest tested dose, while changes in various clinical chemistry parameters were also apparent at this level. Two high-dose females died during the study, each displaying a variety of gross and microscopic effects. The NOAEL for systemic toxicity of lauryl betaine was established as 10 mg/kg bw/day, on the basis of the kidney and bladder histopathology.


Although lower No Observed Adverse Effect Levels were demonstrated by the combined repeated dose/developmental toxicity screening test, the 90-day study (Pittermann, 1993) was chosen as key study, as for regulatory purposes the results of a subchronic study are required. Besides, the applied doses of all studies are within comparable ranges and the test material used for the subchronic study is well comparable to that of the combined repeated dose/developmental toxicity screening test. Therefore, the symptoms described in the latter would have also been expected in the subchronic study, especially in the high dose group, due to the longer study duration. However, histopathology in the study by Whitlow et al. demonstrated irritant effects in the stomach due to repeated gavage of an irritant test material, and in the kidney and bladder due to the excretion of an irritant test article or its metabolites in the urine. Therefore, it is reasonable to consider the effects described in this study, although adverse, as secondary to the observed inflammation reactions.


Dermal:

According to Regulation (EC) No 1907/2006, Annex IX, section 8.6.2, column 2, dermal application is not required, although skin contact has to be anticipated as main route of human exposure. The available human data demonstrate that betaines penetrate human skin only to a minor extent, which is primarily located in the outer layers of the stratum corneum, and appropriate RMMs are already implemented due to the irritant characteristics of the betaines. Workers involved in production of betaines did not show any adverse effects in routine medical check-ups. Data for the oral route are available, additional dermal testing would neither improve risk assessment nor safety of applications.


Inhalation:

According to Regulation (EC) No 1907/2006, Annex IX, 8.6.2, column 2, administration by inhalation is not required. The test substance has a low vapour pressure, exposure to aerosols, particles or droplets is unlikely, and appropriate RMMs are already implemented. Workers involved in production of betaines did not show any adverse effects in routine medical check-ups. Data for the oral route are available, additional inhalation testing would neither improve risk assessment nor safety of applications.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Hazard assessment is conducted by means of read-across based on an analogue approach. The selected study is the most adequate and reliable one based on the identified similarities in structure and intrinsic properties and overall assessment of quality, and with special respect to duration and dose descriptor level (betaine content). DNELs have been derived from the general systemic toxicity NOAEL of 50 mg/kg bw/day reported in the Whitlow (2009) combined repeated-dose/reproductive and developmental toxicity (OECD 422) study. This NOAEL is lower than the LOAEL (of 60 mg/kg bw/day) reported in the JECDB (2005) study by the same method, and so is considered to be suitably health precautionary.

Justification for classification or non-classification

Based on the available information for Beatines, C12 -14 (even numbered)- alkyldimethyl and substances structurally closely related to Betaines, C12-14 (even numbered)-alkyldimethyl, according to the criteria of Regulation (EC) No 1907/2006, Annex XI, section 1.5, those substances do not have to be classified for Specific Target Organ Toxicity - Repeated Exposure according to the criteria of the EU Directive 67/548/EEC and Regulation (EC) No 1272/2008; therefore, Betaines, C12-14 (even numbered)-alkyldimethyl does not have to be classified, either.