Registration Dossier

Administrative data

Description of key information

OECD 408 (Repeated Dose 90-Day Oral): NOAELrat = 145 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
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated 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).
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
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 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 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:

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 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.