Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key 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 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 item 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
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
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
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day (acitve ingredient)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, RCC Study Number B62763.
Positive control:
none
Parental animals: Observations and examinations:
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.

OTHER:
Mating performance and fertility, duration of gestation
Oestrous cyclicity (parental animals):
During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrous cycles.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
The testes and epididymides of all parental males were weighed as pairs. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Litter observations:
STANDARDISATION OF LITTERS
Not performed, pups were sacrificed on day 4.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter size, live births, still births, postnatal loss and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.


GROSS EXAMINATION OF DEAD PUPS:
yes, dead pups were examined macroscopically; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
- Maternal animals: Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®). All P generation animals were exsanguinated.


GROSS NECROPSY
All parent animals were examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system. The number of implantation sites, implantation rate, post-implantation loss, 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 / 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.

The following tissues from all parental males and females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative), Ovaries.

In addition, from the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
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 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. 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 of the low and mid dose groups. 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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age. All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®).
- These pups were examined macroscopically for any structural changes.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations, dead pups were examined macroscopically, as well.
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.
Reproductive indices:
Fertility index/conception rate (percentage of females pregnant), gestation index (percentage of dams not delivering dead pups), mean duration of gestation, implantation rate (number of implantation sites compared to control), Post-implantation loss.
Offspring viability indices:
Litter size at first check, postnatal loss, sex ratio.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females mated within the first mating period. No test item-related effects were noted on the mean precoital time. Two females in the control group were not pregnant (fertility index and conception rate of 80%), as well as 1 female in the low dose group (fertility index and conception rate were 90%). One female in the high dose group gave birth to one dead male pup only, leading to a gestation index at this dose level of 90%. This was considered to be incidental.

The mean duration of gestation was similar in all groups.
The mean number of corpora lutea was not considered to have been affected by treatment with the test item.
There were slightly fewer implantation sites per dam (12.2) in the high dose group compared to the control group (14.8). However, this was within the range of the historical control data and was not statistically significant.
Post-implantation loss was statistically significantly increased (3.0 per dam compared to 0.3 in the control group). This was also within the range of the historical control data but it cannot be excluded that this was a test item-related effect. No test item-related findings were noted in the low and mid dose groups on the implantation rate and post-implantation loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The organ/body weight ratios of the kidneys, liver and adrenals were slightly increased and the thymus slightly decreased in both males and females of the high dose group. In the mid dose group the organ/body weight ratio of the liver was slightly increased in the males and females. In the low dose group no test item-related findings were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the high dose group 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. In the mid dose group a thickened stomach was noted in one male, retractions of the stomach were noted in one female. Foci were found on the stomach of one female in the low dose group.

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

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.
Dose descriptor:
LOEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
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 (bladder, kidney)
Dose descriptor:
NOEL
Remarks:
reproduction/developmental
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 (bladder, kidney)
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
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
In the high dose group, the mean number of pups per litter was statistically significantly reduced (9.2 compared to 14.5 in the control group). This was outside the range of the historical control data. The number of pups per litter in the low and mid dose groups were not considered to have been affected by treatment with the test item.

Postnatal loss was statistically significantly increased (9 in total in 5 litters compared to none in the control group) in the high dose group. In the mid dose group, 3 pups were lost in 2 litters. This was within the range of the historical control data. In the low dose group, no postnatal loss was noted.

CLINICAL SIGNS (OFFSPRING)
No clinical signs were noted at the first litter check or during lactation for any pup in any group

BODY WEIGHT (OFFSPRING)
In the high dose group, the mean body weight of the pups was reduced on day 1 (5.3 grams compared to 5.8 grams in the control group) and was still reduced on day 4 post partum (7.4 grams compared to 8.0 grams in the control group). However, the pups in all groups gained a similar amount of weight over these 4 days. This reduction in body weight was considered to be a test item-related effect.

Mean body weight in the low and mid dose groups was similar to that of the control group.

GROSS PATHOLOGY (OFFSPRING)
In the high dose group, no milk was found in the stomach of 2 female pups in one litter which were already dead at the first litter check. One of these pups had a shortened lower jaw. In another litter, 3 males and 1 female also had no milk in the stomach and were found dead on day 2 post partum. These findings were considered to be incidental.

No other findings were noted at the first litter check or during lactation for any pup in any group.

No abnormal macroscopical findings were noted for any pup in any group at necropsy.

OTHER FINDINGS (OFFSPRING)
On day 4 post partum, the sex ratios were close to 50% in all groups.
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
Dose descriptor:
LOAEL
Remarks:
systemic
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: pup weight
Reproductive effects observed:
not specified

Conclusion:

Although developmental toxicity effects like reduced pup weight, litter size and increased post-implantation and postnatal loss were observed in animals of the highest dose group, these effects have to be considered as secondary to maternal toxicity. Effects observed in parental animals of the highest dose group include sedation, salivation, and irritation effects in the stomach and the bladder due to the irritating nature of the test substance, confirmed by macroscopic and histopathologic findings in the respective organs. These effects were very likely to explain reduced weight gain and reduced absolute body weights and were already observed in less pronounced form in the animals of the mid dose group.

Considering the characteristics and severity of those adverse maternal findings, the observed developmental effects observed in utero and post-partum have to be considered as non-specific and secondary to maternal toxicity.

Therefore, the test substance does not have to be classifed as toxic to reproduction according to the criteria of the DSD and the CLP-regulation.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2, due to read across) from a reference substance with similar structure and intrinsic properties. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, section 8.7, in accordance with Annex XI, section 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are data available from two Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Tests. The first (and its corresponding range-finder) were both performed with the reaction mass of CAS 683-10-3 and CAS 2601-33-4, a substance structurally closely related to Betaines, C12-14 (even numbered)-alkyldimethyl (Whitlow, 2009). The second was conducted on lauryl betaine (CAS 683-10-3), the principal component of ADB (JECDB, 2005).


In the first study (Whitlow, 2009), the animals received doses of 50, 150 and 300 mg/kg bw/day, referring to active ingredient of the reaction mass, by oral gavage. Males were treated for a minimum of 4 weeks (28 days), then they were sacrificed, while females were treated for approximately 7 weeks (49 days), they were sacrificed on day 5 postpartum. In males, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell cultures. In females, the numbers of implantation sites, implantation rate, post-implantation loss and corpora lutea were recorded for all dams with litters. Although developmental toxicity effects like reduced pup weight, litter size and increased post-implantation and postnatal loss were observed in animals of the highest dose group at 300 mg/kg bw/day, these effects have to be considered as secondary to maternal toxicity. Systemic effects observed in parental animals of the highest dose group included sedation, salivation, and irritation effects in the stomach and the bladder due to the irritating nature of the test substance, confirmed by macroscopic and histopathologic findings in the respective organs. These effects were very likely to explain reduced weight gain and reduced absolute body weights and were already observed in less pronounced form in the animals of the mid dose group at 150 mg/kg bw/day. Considering the characteristics and severity of those adverse maternal findings, the observed developmental effects observed in utero and post-partum have to be considered as non-specific and secondary to maternal toxicity.


In the corresponding range-finder study (Whitlow and Flade, 2008) only 3 animals were included per dose group. Male and female animals received doses of 33, 100, 300 and 1000 mg/kg bw/day, referring to active ingredient of the reaction mass, by oral gavage for 28 and 42 days, respectively, and were assessed for clinical signs, food consumption and body weight. Males were sacrificed and necropsied after completion of the pairing period, females on day 14 post coitum. For evaluation of fertility, the number and distribution of implantation sites, corpora lutea, live or dead embryos, and early and late embryonic deaths were recorded. Animals receiving 300 mg/kg bw/day showed signs of systemic toxicity like sedation and reduced body weights, body weight gain and food consumption. No effects on fertility parameters were observed at that dose. No animals had survived for assessment at 1000 mg/kg bw/day, all animals in this dose group died within 24 hours after the first treatment.


In the OECD 422 study of lauryl betaine, the test substance was administered daily by oral gavage at doses of 10, 60 and 300 mg/kg bw/day. Males were treated for 42 days pre-mating and throughout the mating period [approximate total duration of treatment, 56 days]. Females were treated for 2 weeks before mating, and throughout the mating, gestation and lactation periods, and were killed 4 days post-partum [approximately 53 days]. Control animals received vehicle only. In the high-dose group, decreased number of births, decreased fertility rate and prolonged gestation period were observed, as well as one case of the death of all pups on the first day of lactation. There was no difference between treated and control animals in terms of mating, conception or implantation rates, or of pup weight, condition or survival rate. On this basis, the NOAEL for reproductive and developmental toxicity of lauryl betaine was established as 60 mg/kg bw/day.No substance-specific data is available on the toxicity to reproduction for Betaines, C12-14 (even numbered)-alkyldimethyl. Nevertheless, due to the similarities in the composition of the two source substances in the above studies (as further discussed in the read-across justification report attached to Section 13), further studies on the endpoint "toxicity to reproduction" are not required, read-across from both source susbtances is justified and represents an acceptable approach to satisfy the data requirements for the endpoint.


According to Regulation (EC) No 1907/2006, Annex IX, section 8.7.3. column 1, a 2-generation study for assessment of reproductive toxicity is not required as no adverse effects on reproductive organs were reported in the subchronic 90-day study.


Short description of key information:

Read-across from reaction mass of CAS 683-10-3 + CAS 2601-33-4: Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (OECD 422) in rats: NOAELfertility = 150 mg/kg bw/day (in presence of maternal toxicity); NOAELsystemic (parental) = 50 mg/kg bw/day


Data waiving - Two-generation reproductive toxicity study


Justification for selection of Effect on fertility via oral route:

Hazard assessment is conducted by means of read-across based on an analogue approach. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

Read-across cocamidopropyl betaine: Prenatal developmental toxicity (OECD 414), rats: NO(A)ELmaternal = 100 mg/kg bw/day; NO(A)ELembryotox = 300 mg/kg bw/day; NO(A)ELteratogenicity ≥ 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-10-22 to 2004-02-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 22, 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Non-clinical Studies of Drugs Manual 1995; Guidelines for Toxicity Studies of Drugs. Japanese Ministry of Health and Welfare.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
certificate Stadt Hamburg, Germany
Limit test:
no
Species:
rat
Strain:
other: CD/Crl:CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Age (at day 0 of pregnancy): 8 - 9 weeks
- Weight (at day 0 of pregnancy): 205 - 254 g
- Fasting period before study: none
- Housing: singly in MAKROLON cages
- Diet: ad libitum, ssniff R-Z V1324, ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water : ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/-2 °C
- Humidity: relative 55% +/- 15%
- Photoperiod: 12 hours dark/12 hours light, 150 lux at app. 1.5 m room height

IN-LIFE DATES: From: Oct 22, 2003 To: Nov 19, 2003
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Control: 10 ml vehicle/kg bw /day
Dose levels: The dose levels referring to active ingredients were nominal 100, 300, and 1000 mg/kg bw/day and referring to test item 330, 990 and 3300 mg/kg bw/day. The nominal concentrations were analytically verified in samples taken at study initiation and study termination. The actual concentrations of the samples taken from the aqueous test item carrier mixtures were within the range of 101.9% to 109.9% of the nominal concentrations indicating correctly prepared application mixtures and a sufficient stability.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqua ad iniectabilia
- Lot/batch no. (if required): 3175P13E/F, B. Braun Melsungen AG, D-34212 Melsungen, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the concentration of the test item-carrier mixtures, samples of approx. 10 mL were taken at the following time-points and stored at -20°C or colder until analysis by LPT:
- At study initiation (1 st administration): Concentration and stability immediately after preparation of the mixture as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). total number of samples: 9; Homogeneity at start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). total number of samples: 9
- At termination of the administration period: Concentration during treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). total number of samples: 3
The method used was made available by the sponsor and re-validated by LPT. Analytical Method for the Determination of C8-18 AAPB in Water with UV/VIS Detection. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity, stability.
The results of the analyses showed that the test item-carrier mixtures were correctly prepared and the concentration and stability found were in good agreement to those expected. The actual concentrations of the test item-carrier mixtures were within the measured range of 101.9% to 109.9% of the nominal C8-18 AAPB concentrations.

Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. They were repeatedly employed, at the earliest two days after successful copulation. Females mated by the same male were placed in different groups (if possible). The female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm and the stage of oestrus cycle. If findings were negative, mating was repeated. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
14 days, from 5th to the 19 th day of pregnancy, the day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
Frequency of treatment:
daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw /d
Basis:
analytical conc.
based on a.i.
No. of animals per sex per dose:
Mated and treated animals: group 1 - 4: 25
Evaluated pregnant rats: group 1 - 4: 20 (the first 20 animals with pregnancy signs were used)
Animals evaluated for maternal toxicity: group: 1, 2, and 3: 20, group 4: 21 (one additional animal was included due to the premature death of one high-dosed dam)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on available toxicity data of a former 14-day dose-range-finding to determine suitable dose
levels for a subsequent 90-day toxicity study.
- Rationale for animal assignment (if not random): the rat is a commonly used rodent species for embryotoxic studies
- Other: The test item was delivered as a 30% aqueous solution as it is marketed. To specify the impact of C8-18 AAPB the doses are calculated on the active ingredient. Whilst preparing the dosing solution a correction factor of 3.3 was used. Hence all dosing levels in this study refer to the active ingredient.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
-- Viability checks in addition to detailed clinical observation were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
-- Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on day 0 of gestation, followed by daily weighings, always at the same time and carcass weight, once at termination
-- The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. 0-3, 3-6, 6-9, 9-12, 12-15,15-18 and 18-20). Furthermore the net weight change from day 6 was calculated (= carcass weight minus day 6 body weight; whereas carcass weight = terminal body weight minus uterine weight).
These measurements were also used for calculating the daily amount of test item to be administered.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-- The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day. The relative food consumption (g/kg bw/day) was calculated using the following formula: Daily food consumption [g/kg bw/day] = Total food intake in g/Body weight in g * 1000

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily
-- Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries, uteri, internal organs, placentae
-- On the 20th day of gestation, the rats were laparotomised under ether narcosis. The ovaries and uteri were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations. Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations:
- The number of fetuses (alive and dead) and placentae was determined.
- Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
- Number and size of resorptions were determined.
- Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
- The gravid uterus weight was determined.
- Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
- Fetuses were inspected externally for damages, especially for malformations
The fetuses were sacrificed by an ether atmosphere.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
-- 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
- Skeletal examinations: Yes
-- 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
Statistics:
Bartlett chi-square test for: numerical values, homogeneity of variances
Dunnet test: when variances were homogeneous, test was used to compare the experimental groups with control groups
Students test: in case of heterogeneity of variances
Fisher´s exact test: for comparison of classification measurements (i.g. malformation-, resorption-, retardation, and variation rate)

Indices:
- Corpora lutea: number per dam, absolute number per group, mean per group
- Implants: number per dam, distribution in the uterine horns, absolute number per group, mean per group
- Resorptions: number per dam, distribution in the uterine horns, absolute number per group, mean per group, mean % per group, early resorptions < 2mm, late resorptions > 2 mm
- Resorption rate [%] = (resorptions/implantations) x 100
- Weight of placentae: individual data per fetus, mean per litter, mean per group, litter mean per group, litter mean per sex and group
- Weight of fetuses: individual data per fetus, mean per litter, mean per sex and litter, litter mean per group, litter mean per sex and group
- Fetuses:number per dam (alive and dead), number of fetuses per sex and dam, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group
- Dead fetuses: number per dam, mean per group
- Runts: number per dam, mean per group
- Malformed fetuses: individual data per fetus, mean per group and type of malformation
- Malformation rate per group [%] = (malformed fetuses/fetuses) x 100
- Fetuses with variations: individual data per fetus, mean per group and type of variation
- Variation rate per group [%] = (fetuses with variations/fetuses) x 100
- Fetuses with retardations: individual data per fetus, mean per group and type of retardation
- Retardation rate per group [%] = (fetuses with retardations/fetuses) x 100
- Pre-implantation loss [%] = ((corpora lutea - implantations)/corpora lutea) x 100
- Post-implantation loss [%] =((implantations - living fetuses)/implantations) x 100

Historical control data:
Summarized results of the 19 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at LPT in the years 2000 - 2003 were used as historical control data. These background data have not been audited by the Quality Assurance Unit of the testing facility.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced food consumption, impaired body weight and necropsy findings at 300 and 1000 mg a.i./kg bw/day
For further details see section "Any other information on results including tables "
Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Reduced fetus weight and increased number of resorptions at 1000 mg a.i./kg bw/day
For further details see section "Remarks on results including tables and figures"
Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
MATERNAL TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:Mortality: 100 and  300 mg a.i./kg bw / day: no mortality1000 mg a.i. /kg bw / day: 1/20 on gestation day 15

Clinical signs:

100 and 300 mg a.i./kg bw / day: no clinical signs of systemic toxicity

1000 mg a.i./kg bw /day: an abdominal position was noted in 13 of 21 dams on several (1 to 6) gestation days starting from the second administration day (gestation

day 6). This symptom started within 5 to 20 minutes after dosing and lasted for 20 minutes to 2 hours. Moreover, pilo-erection and reduced motility were noted in two

dams of the high dose group. This symptom was noted in one dam on gestation days 19 and 20 and in dam which died on gestation days 12 to 15 (until premature death).

The faeces of all dams were of normal consistency during the whole experiment..

Body weight:

100 and 300 mg a.i./kg bw / day: no test item-related influence on the body weight. A marginal, but statistically significant decrease by 4% to 5% was noted in the low

dose group (100 mg/kg bw / day) on gestation days 10 to 17 for the body weight. This is regarded to be spontaneous due to no comparable effects at 300 mg/kg

bw / day.

1000 mg a.i./kg bw / day: the body weight was moderately reduced (up to 17% below the control values, significant at p </= 0.01) from the start of treatment onwards until

laparotomy on gestation day 20.

Body weight change:

100 and 300 mg a.i./kg bw / day: No test item-related influence was noted on the body weight change.

1000 mg a.i./kg bw /day: the mean maternal body weight change was statistically significantly (at p </= 0.01) decreased on gestation days 3 to 6, 6 to 9, 12 to 15, 15

to 18 and 18 to 20.

Food and drinking water consumption:

100 mg a.i./kg bw / day: The food consumption was not influenced.

300 mg a.i./kg bw / day: The food consumption was marginally reduced on some days during the administration period (up to 12%, statistically significant on gestation days

8,10 and 19 at p </= 0.05 or p </=0.01).

1000 mg a.i./kg bw / day: A severely and statistically significantly (at </= 0.01 or p </= 0.05) reduced food consumption was noted from start of treatment until laparotomy,

predominantly during the first treatment days.

Drinking water consumption showed no test item-related changes in any of the treated groups as observed during daily visual appraisal.

EXAMINATION OF THE DAMS AT TERMINATION

Necropsy findings Stomach:

100 mg a.i./kg bw / day: No test item-related pathological findings.

300 mg a.i./kg bw / day: Thickened/partly thickened stomach mucosa in 4 of 20 animals, in addition ulcers (diameter approximately 1 mm or 0.5 to 1 mm) in 2/4 animals with thickened mucosa.

1000 mg a.i./kg bw /day: Thickened or partly thickened stomach mucosa (greyish discoloured in two dams) was noted in 20 of 21 dams including one prematurely deceased dam. In addition, in two of these dams a few ulcers were noted in the stomach (diameter up to 1 mm). These findings are regarded to be test item-related.

Necropsy findings other:

A reduced in size spleen was noted in one high-dosed dam (1000 mg/kg b.w./day) and is regarded to be an incidental finding.

Gravid Uterus weight:

100 and 300 mg a.i./kg bw / day: No test item-related influence.

1000 mg a.i./kg bw /day: A reduction by 22% (significant at p </= 0.01) was noted for the gravid uterus weight caused by the lowered fetal weights.

Carcass weight:

100 and 300 mg a.i./kg bw / day: No test item-related influence.

1000 mg a.i./kg bw / day: The carcass weight was statistically significantly (at p </= 0.01) reduced by 15% when compared with the control animals

Net body weight change from day 6 (= carcass weight minus day 6 body weight):

100 mg a.i./kg bw / day: No test item-related influence.

300 and 1000 mg a.i./kg bw / day:a statistically significant (at p </= 0.05 or p </= 0.01) reduction by 23% and 67%, respectively, was noted for the net weight change from day 6 onwards.


REPRODUCTION DATA OF DAMS (0, 100, 300, 1000 mg/kg bw):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg a.i. /kg bw / day with respect to the number of corpora lutea and implantation sites.

The number of resorptions (early, late and total) was increased in the high dose group (1000 mg/kg bw / day). Moreover, a statistically significant (p </= 0.01) increase was noted for the ratio of early, late and total resorptions to implantation sites in this dose group. As a result, the number of viable fetuses and the ratio of viable fetuses to implantation sites (statistically significant at p </= 0.01) were decreased at 1000 mg a.i. /kg bw / day. These changes are caused by a total post-implantation loss in two dams at an early time of pregnancy and are regarded to be test item related. However, the effects have to be considered as secondary due to maternal toxicity.

Detailed indices for test groups 0 (control), 100, 300 and 1000 mg a.i./kg bw / day, respectively:
- Corpora lutea: 301 (15.1 per dam), 313 (15.7 per dam), 311 (15.6 per dam), 316 (15.8 per dam)
- Implantation sites: 290 (14.5 per dam), 309 (15.5 per dam)*, 309 (15.5 per dam)**, 307 (15.4 per dam)
- Resorptions: 10 (0.5 per dam), 7 (0.4 per dam), 12 (0.5 per dam), 53 (2.7 per dam)**
- Early resorptions: 10 (0.5 per dam), 3 (0.2 per dam)*, 9 (0.5 per dam), 46 (2.3 per dam)**
- Late resorptions: 0 (0.0 per dam), 4 (0.2 per dam)*, 3 (0.2 per dam), 7 (0.4 per dam)**
- Live fetuses: 280 (14 per dam), 302 (15.1 per dam), 297 (14.9 per dam), 254 (14.1 per dam **, n= 18 dams with visible fetuses)

- Dead fetuses at laparotomy: in all test groups 0

- Pre-implantation loss (mean %): 6.3, 1.1, 0.6, 2.8

- Post-implantation loss (mean %): 3.3, 2.3, 4.2, 17.5

* Significantly different from control, p </= 0.05
** Significantly different from control, p </= 0.01

- comparing the ratio of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group

- comparing the ratio of resorptions/implantation sites of the test group with the ratio of resorptions/implantation sites of the control group

- comparing the ratio of fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group

EXAMINATION OF THE FETUSES:

Sex distribution of fetuses:

100, 300 and 1000 mg a.i./kg bw / day: The sex distribution of the fetuses was comparable with that of the control fetuses.

Weight of placentae:

100, 300 and 1000 mg a.i./kg bw / day: The mean placental weights were not influenced by the administration of the test item to the dams when

compared with the control group.

Weight of fetuses:

100 and 300 mg a.i. /kg bw / day: The mean fetal weights were not influenced as compared to the control group.

1000 mg a.i./kg bw / day : The mean fetal weights calculated for male and female fetuses and for all fetuses were statistically significant (at p </= 0.01) below the control values. Although the mean fetal value of this group is still within the range of LPT background data, this effect is regarded to be test item-related. However, the effect has to be considered as secondary due to maternal toxicity.

External examination of the fetuses:

100, 300 and 1000 mg a.i./kg bw / day: No macroscopically visible malformations and variations were noted during external examination at laparotomy.

Laparotomy revealed no dead fetuses at any tested dose level. One runt each was noted at 100 and 300 mg/kg b.w./day. This change is regarded

to be spontaneous and is within the normal range of variation.

Skeletal examination of the fetuses:

100, 300 and 1000 mg a.i./ kg bw / day: The skeletal examination (according to DAWSON) revealed no malformed fetuses at any of the tested dose level and in the control group.

- The skeletal variations observed were related to the ribs (accessory 14th rib(s), rib(s) not ossified, shortened or wavy) and the sternum (sternebra(e) bipartite, dumbbell shaped or misaligned to a slight degree).

- No test item-related skeletal variations were noted at 100, 300 or 1000 mg a.i./kg bw / day.

- Although there was no statistical significance for the incidence of each variation in either dose group, a slight but statistically significant increase (at p </= 0.05) was seen in the total incidence of skeletal variations at 300 mg a.i./kg bw / day. This finding was judged as incidental as no dose-relationship was noted.

- Skeletal retardations were related to the 5th metacarpalia (not ossified), caudal vertebral bodies (only one body ossified), lumbar vertebral body/bodies (less than 6 ossified, bipartite), thoracic vertebral body/bodies (bipartite, dumbbell-shaped), hyoid (missing ossification), skull (incomplete ossification), sternebra(e) (incomplete or missing ossification, reduced in size).

- No test item-related influence was noted for the incidence of skeletal retardations at any of the tested dose levels (100, 300 or 1000 mg a.i./kg bw / day).

- Increased fetal and litter incidences (significant at p </= 0.05 or p </= 0.01) observed for not ossified 5th metacarpalia in all dosed groups are related to the low value obtained for the control group. Further, the observed incidences are still within the LPT background data of 0% to 7.8% mean fetal incidence for this retardation type.

- All other significances noted in the test item groups (fetal incidences for not or incompletely ossified hyoid, skull or sternebrae as well as fetal incidence for the total fetal skeletal retardations) are regarded to be without biological relevance, as these changes refer to a decrease in comparison with the control group.

Soft tissue examination of the fetuses:

100, 300 and 1000 mg a.i./kg bw / day: The examination of the fetal organs (according to WILSON) revealed no malformed fetuses at any of the tested dose level and in the control group.
- The fetal examination according to WILSON revealed the following variations: 4th cerebral ventricle dilated, cardiomegaly, dilated renal pelvis, misplaced kidney and
haemorrhage / haemorrhagic focus/foci in the liver or thoracic cavity. No statistically significant differences in fetal or litter incidences were noted for these variations at any of the tested dose levels (100, 300 or 1000 mg/kg b.w./day). These findings are very common in the rat strain used and the incidences observed were within the historical background range.

Detailed indices for test groups 0 (control), 100, 300 and 1000 mg a.i./kg bw / day, respectively:

- Malformations (external, skeletal, soft tissue) (fetal incidence): in all test groups 0
- External variations (fetal incidence): in all test groups 0
- Skeletal variations (fetal incidence):5, 8, 13*, 6 (finding was judged as incidental as no dose-relationship was noted)
- Skeletal retardations (fetal incidence): 129, 137, 130, 125
- Soft Tissue variations (fetal incidence): 8, 12, 10, 9

* Significantly different from control, p </= 0.05


Conclusions:
In this developmental toxicity / teratogenicity study, performed according to OECD TG 414 on CD rats, 330, 990 and 3300 mg/kg bw/day of a 28.9 % aqueous C8-18 AAPB solution, corresponding to 100, 300, and 1000 mg active substance/kg bw/day, respectively, were applied by gavage. Dose-related maternal toxic effects (reduced food consumption, impaired body weight and necropsy stomach findings) occurred at 990 mg/kg bw/day and above. Embryotoxic effects (reduced mean fetal weight and increased number of resorptions) were found only at the maternal toxic dose level of 3300 mg/kg bw/day. Up to and including the highest tested dose, no external, skeletal or soft tissue malformations and no external variations were found. The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active substance/kg bw/day) and the NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active substance/kg bw). The NOEL for teratogenic effects was the highest tested dose of 3300 mg/kg bw/day, corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.
Executive summary:

In a developmental toxicity study according OECD 414, C8 -18 AAPB (28.9 % a.i, 62 % water, and 5.4 % NaCl) was administered to 25 females CD rats/dose at dose levels of 0, 330, 990, 3300 mg from day 5 through 19 of gestation by gavage. The test item dose levels refer to nominal active ingredient of 100, 300, and 1000 mg/kg bw/day. The nominal values were analytically verified in samples taken at study initiation and study termination. The actual concentrations of the samples taken from the aqueous test item carrier mixtures were within the range of 101.9% to 109.9% of the nominal C8 -18 AAPB concentrations indicating correctly prepared application mixtures and a sufficient stability. Number of evaluated pregnant rats were 20/group (the first 20 animals with pregnancy signs were used). Animals evaluated for maternal toxicity were 20/group except of high dose group in which one additional animal was included due to a premature death of one dam.

Regarding maternal toxicity, the dams of the 990 mg/kg bw/day group showed decreased net body weight change from day 6 onward (= carcass weight minus day 6 body weight), reduced food consumption, thickened/partly thickened stomach mucosa in 4 of 20 animals and in addition ulcers (diameter approximately 1 mm or 0.5 to 1 mm) in 2/4 animals with thickened mucosa. In the 3300 mg/kg bw/day group the dams showed severely reduced food consumption, reduced body weights (absolute, body weight gain on gestation days 3 to 6, 6 to 9, 12 to 15, 15 to 18 and 18 to 20, and net body weight change from day 6 onward), reduced carcass weight and reduced gravid uterus weights. Thickened or partly thickened stomach mucosa (greyish discoloured in two dams) was noted in 20 of 21 dams including one prematurely deceased dam. In addition, in two of these dams a few ulcers were noted in the stomach (diameter up to 1 mm).

The number of early, late and total resorptions was increased in the 3300 mg/kg bw/day group, and the ratio of viable fetuses to implantation sites was decreased compared to the controls. This was due to a total post-implantation loss in two dams in this dose group. In addition, a statistically significant reduction in fetal weights and in the number of viable fetuses as compared to the control was observed. No external, skeletal or soft tissue malformations and no external variations were found. The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active ingredient/kg bw/day). The NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active ingredient/kg bw/day).

The NOEL for external, skeletal or soft tissue malformations and variations was the highest tested dose of 3300 mg/kg bw/day (corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study in rat.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex IX, section 8.7.2, in accordance with Annex XI, section 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There were no appropriate substance-specific data available for Betaines, C12-14 (even numbered)-alkyldimethyl. Therefore, data from structurally and chemically related test substances (according to the criteria set out in Regulation (EC) No 1907/2006, Annex XI, section 1.5) have been used for assessment of developmental toxicity in rats. Two studies (Hoberman and Christian, 1984; Arnold et al., 1985) were conducted on cetyl betaine, a close structural analogue and minor component of the target substance. A third study tested cocamidopropyl betaine (C8-18 AAPB; CAPB) (CEFIC/CESIO [ICCA Initiative] Alkylamidopropyl Betaines Consortium, 2004). Insights on developmental toxicity are also available from a combined repeated-dose toxicity with reproductive/developmental toxicity screening study, conducted on lauryl betaine, the principal component of ADB (JECDB, 2005).


The pre-natal developmental toxicity study of cetyl betaine (Hoberman and Christian, 1984), in apparent accordance to US EPA Test Guideline 870.3800, was the only relevant study identified to be conducted in rabbits. Cetyl betaine was topically applied (without covering) to the skin of pregnant New Zealand White does (8/group) at doses of 2, 10, 20, 40, 100 or 200 mg/kg bw/day, for 4 hours/day on gestation days 6-18. The test site was rinsed at the end of each treatment period. Control animals received vehicle only. Due to test item-related maternal mortality and the severity of local irritant effects, the 100 and 200 mg/kg bw/day groups were terminated early, after the 8th and 6th applications, respectively. Average body weight gain and average daily feed consumption were reduced in all treated animals, and reduced to a biologically significant degree at and above 10 and 40 mg/kg bw/day, respectively. The resorption rate was also increased from 40 mg/kg bw/day upwards, with an associated decrease in average litter size at the two highest dose levels. All foetuses were alive at maternal Caesarean-section, but no further examination was conducted. The EPA’s summary of this study reported a developmental toxicity NOAEL of 40 mg/kg bw/day, while in contrast, the CIR Panel reported a NOAEL of 20 mg/kg bw/day.


The developmental toxicity of cetyl betaine has also been assessed in rats, again following US EPA Test Guideline 870.3800 (Arnold et al., 1985). Pregnant dams were administered the test material by oral gavage at doses of 0, 50, 150 or 250 mg/kg bw/day, on gestation days 6-15. Control animals received vehicle only. No mortality occurred in control or treated dams and there were no treatment-related differences upon necropsy. However, a dose-dependent reduction in maternal growth and food intake was noted in all treatment groups. At the highest tested dose (250 mg/kg bw/day), there was as an increased incidence of litters with reduced or absent ossification of the skull and sternebrae. Fetal viability and body weights were unaffected by treatment. The NOAEL for developmental toxicity of cetyl betaine was established as 150 mg/kg bw/day, while the maternal systemic toxicity LOAEL was 50 mg/kg bw/day.


In the study of CAPB, pregnant rats were treated daily by oral gavage from Gestational Day 5 to 19 with test substance doses of nominal 0, 100, 300 and 1000 mg/kg bw/day, referring to active ingredient. Signs of maternal toxicity manifested as mortality (1/20) at the high dose and as local irritation in form of thickening of the gastric mucosa and ulcers in the mid and high dose dams, found upon necropsy. Additionally, reduced body weight and reduction of food consumption were observed, both marginal in the mid dose group and moderate and severe, respectively, in the high dose group (CEFIC/CESIO, 2004).


Developmental toxicity was observed as reduced fetal weight and an increased number of resorptions in the high dose group. These observations were considered to be test substance-related, but have to be regarded as typical secondary effects due to maternal toxicity. No test item-related skeletal retardations or soft tissue malformations were observed in any of the dose groups. Therefore, a NO(A)EL of 100 mg/kg bw/day was established for maternal toxicity; for embryotoxicity a NO(A)EL of 300 mg/kg bw/day was derived. The NO(A)EL for teratogenicity was determined to be 1000 mg/kg bw/day, the highest tested dose and corresponding to the guideline limit dose (CEFIC/CESIO, 2004).


In the OECD 422 study of lauryl betaine, the test substance was administered to rats daily by oral gavage at doses of 10, 60 and 300 mg/kg bw/day. Males were treated for 42 days pre-mating and throughout the mating period [approximate total duration of treatment, 56 days]. Females were treated for 2 weeks before mating, and throughout the mating, gestation and lactation periods, and were killed 4 days post-partum [approximately 53 days]. Control animals received vehicle only. The critical effects at the high-dose level were a decrease in the number of births and one case of the death of all pups upon delivery (possibly as a result of the observed dystocia in the dam). There was no difference between treated and control animals in terms of pup weight, condition or survival rate. Maternal toxicity in the form of microscopic effects on the kidney and bladder (also observed in males) was evident at and above 60 mg/kg bw/day. On this basis the NOAEL for maternal toxicity of lauryl betaine was established as 10 mg/kg bw/day, while the NOAEL for developmental toxicity was considered to be 60 mg/kg bw/day (JECDB, 2005).


Justification for selection of Effect on developmental toxicity: via oral route:

Hazard assessment is conducted by means of read-across based on an analogue approach. The selected study is adequate and reliable based on the identified similarities in structure and intrinsic properties and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details). In addition, no other data sources are available for this endpoint.

Justification for classification or non-classification

Based on the available information for a substance 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, that substance does not have to be classified for toxicity to reproduction 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.