1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- subacute (28 d) repeated dose toxicity study with additional focus on reproductive organs, oral (gavage), Sprague-Dawley rat, m/f; OECD TG 407; GLP; RL1, dose levels: 0, 100, 300, 500 mg a.i./kg bw/d; NOAEL = 500 mg/kg bw/d
- subchronic (90 d) repeated dose toxicity study with additional focus on reproductive organs, oral (gavage), Sprague-Dawley rat, m/f; OECD TG 408; GLP; RL1, dose levels: 0, 75, 150, 300 mg a.i./kg bw/d; NOEL = 300 mg/kg bw/d; read-across: C8-18 AAPB
- subchronic (90 d) repeated dose toxicity study with additional focus on reproductive organs, oral (diet), Wistar rat, m/f; OECD TG 408; GLP; RL1, dose levels: 0, 9.5, 24, 97 and 247 mg a.i./kg bw/day/d; NOEL = 247 mg/kg bw/d; read-across: C8-18 and C18 unsatd. AAPB
- subchronic (90 d) repeated dose toxicity study with additional focus on reproductive organs, oral (gavage), Wistar rat, m/f; OECD TG 408; GLP; RL1, dose levels: 0, 100, 300, 1000 mg a.i./kg bw/day; NOAEL = 1000 mg/kg bw/d; read-across: Formamidopropylbetaine

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

Repeated Dose 28-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-08-21 to 2015-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 27-29 days
- Weight at study initiation: 92-100 g for males and 80-95 g for females at arrival
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polisulphone solid bottomed cages; nesting material was provided inside suitable bedding bag and changed at least twice a week
- Diet (e.g. ad libitum): powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approx. 3 wk

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

Route of administration:

oral: gavage

Vehicle:

other: purified water (softened water by reverse osmosis)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The formulations were prepared daily at concentrations of 10, 30 and 50 mg/mL. Concentrations were calculated and expressed in terms of test item corrected against the declared purity.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration) and a 28 hour and 8 day stability at room temperature was verified in the same range, to confirm that the method was suitable and stability was satisfactory.
Final results for all levels were within the acceptability limits for concentration (90-110%).
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the concentration. Results of the analyses were within the acceptability
limits for concentration of solutions (90-110%)

Duration of treatment / exposure:

4 weeks + recovery period of 2 weeks

Frequency of treatment:

daily

Details on study schedule:

-animals were not mated

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 male and 5 female; control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results with similar substances
- Rationale for animal assignment (if not random): computerised stratified randomisation to give approximately equal initial group mean body weights
- Rationale for selecting satellite groups: to assess recovery from any treatment-related effects
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

n.a.

Parental animals: Observations and examinations:

MORTALITY: Yes
- twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, approximately 1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once per week from the start of treatment
- parameters: gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern)

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly starting from the day of allocation to treatment group and just prior to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER: NEUROBEHAVIOURAL EXAMINATION, CLINICAL CHEMISTRY, HAEMATOLOGY
- described in more detail in section "Repeated dose toxicity"

Oestrous cyclicity (parental animals):

The histopathological evaluation of the oestrous cycle in the uterus/ cervix and vagina from all animals in the control and high dose groups of the
main phase was performed.

Sperm parameters (parental animals):

The testes and epididymides of main group animals were cut at 2-3 µm thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 4 weeks of treatment.

Litter observations:

n.a.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Postmortem examinations (offspring):

n.a.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.
If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Reproductive indices:

n.a.

Offspring viability indices:

n.a.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were seen during the study.
No toxicological relevant changes were observed at the weekly detailed clinical signs.
Statistically significant decreases in rearing were seen in males receiving 300 and 500 mg/kg bw/day during Weeks 3 and 4 of treatment, increases were seen in females receiving 100 and 300 mg/kg bw/day during Week 3 and in females receiving 500 mg/kg bw/day during Week 4 of treatment, when compared to controls. Since the direction of changes was opposite in the two sexes, the above mentioned changes were considered incidental and not treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in body weight were seen between treated and control animals during the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences in food consumption were seen between treated and control groups during the study.

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted. All observed changes in organs and tissues are considered as incidental-age related findings, characteristically seen in untreated Sprague Dawley rats of the same age or comparable with control animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

fertility parameters

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects up to and including the highest tested dose level

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects up to and including the highest tested dose level

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

no

Conclusions:

On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL for general toxicity and the NOEL for fertility parameters.

Executive summary:

In a subacute toxicity study according to OECD guideline 407 (2008) and EU method B.7 (2008) C8-10 Alkylamidopropyl betaine (34.65% a.i.) was administered to 5Hsd: Sprague Dawley SDrats/sex/dose in purified water by gavage at dose levels of 0 (control), 100, 300 and 500 mg/kg bw/day for 28 consecutive days. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Neurotoxicity assessment did not reveal any treatment-related effects. No changes on body weight and food consumption were noted.

The lymphocytosis and monocytosis seen in single females dosed at 300 and/or 500 mg/kg bw/day showed reversibility at the end of the recovery period or comparability to control data. No toxicological relevant effects in coagulation and clinical chemistry parameters were observed. No differences were reported in terminal body weights and organ weights between treated and control animals and no treatment-related changes were noted at macroscopic and microscopic observations.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL (No Observed Adverse Effect Level).

The NOEL for fertility parameters is 500 mg/kg bw/d.

Reference 2

Endpoint:

fertility, other

Remarks:

assessment of reproduction organs during Repeated Dose 90-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Colworth Wistar
- Age at study initiation: 4-6 weeks
- Weight at study initiation (g): males: 132.9 - 138.3, females 101.7 to 104.5 (group mean values)
- Fasting period before study:
- Housing: singly housed in stainless steel or galvanised cages with stainless steel mesh floors awl doors, such that there was one animal per cage; 48 (four rows x six columns) cages were accommodated on each holding battery.
- Diet: ad libitum, MODAIN purified diet
- Water: e.g. ad libitum, potable tap water from the local water mains
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2
- Humidity (%): 55 +/-10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: MODAIN purified diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): most probably every other week (7 batches in the course of the 13 week study were prepared)
- Mixing appropriate amounts with (Type of food):
-- The formulation of the MODAIN purified diet (%) was Maize starch 65.0 %, Casein (spray dried) 20.0%, Solkafloc 5.0 %, Maize oil 5.0%, Modified AIN-76 minerals 3.5 %, AIN-76A vitamins 1.0 %, DL methionine 0.3 %, Choline bitartrate 0.2 %.
-- The test item was added to the diet at the expense of maize starch for the 0.40% and 1.00% dietary levels. The 1.00% diet was used to prepare the 0.10% dietary level by a 1:10 dilution with control MODAIN purified diet. Similarly the 0.40% diet was used to prepare the 0.04% dietary level by a 1:10 dilution with control MODAIN purified diet
- Storage temperature of food: cold

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets prepared for feeding at the beginning (Batch 1), middle (Batch 4) and end of the study (Batch 7) were sampled and analysed for test item to confirm dietary concentration, and for moisture, protein, fat, fibre and ash to confirm composition. Control MODAIN purified diet is routinely analysed for nutrients and contaminants. For confirmation of dietary concentration, the test item was extracted from the diet samples by sochlet extraction with a methanol: ammonia (100:1) mixture. The extracts were concentrated and analysed by HPLC on a 3µ Nucleosil C18 column using a mobile phase of methanol: water: ammonia (90:10:1) at a flow rate of 0.8 ml/min with detection by a light scattering detector. Quantitation was achieved by comparison of sample peak heights with those of external standards in the range 0.3 - 1.5 mg/mL.
Homogeneity and stability of the test substance within the diets were determined on a 5 kg batch of each diet containing (A) 1.00%, (B) 0.40% and (C) 0.10% test item (the 0.04% dietary level would be covered by the fact that it was prepared by a 1:10 dilution of the 0.40% dietary level with control MODAIN purified diet). Each batch was sampled at 5 positions in the mixing bowl as follows: A top centre, B middle centre, C bottom centre, D left centre and E right centre. Duplicate analyses were completed on each sample after it was prepared. The remainder of each batch of diet was divided into 2 parts, which were stored in an animal room (21 ± 2°C) or a cold room (4°C). Samples were taken for analysis after 7 and 14 days storage. Recovery tests were also carried out on diets spiked with the test item at levels covering the range of incorporation of the test item in the diet.

Duration of treatment / exposure:

mains study: 13 weeks
recovery period: further 32 days

Frequency of treatment:

daily

Details on study schedule:

animals were not mated

Dose / conc.:

0.04 other: % in diet

Remarks:

corresponds to 9.5 mg a.i./kg bw/day

Dose / conc.:

0.1 other: % in diet

Remarks:

corresponds to 24 mg a.i./kg bw/day

Dose / conc.:

0.4 other: % in diet

Remarks:

corresponds to 97 mg a.i./kg bw/day

Dose / conc.:

1 other: % in diet

Remarks:

corresponds to 247 mg a.i./kg bw/day

No. of animals per sex per dose:

main study groups: 12 males and 12 females per group
additional recovery groups: 5 males and 5 females (control and high dose group)

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice each day (once on Saturdays and Sundays) for signs of ill-health and reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals vere given a detailed examination twice a week.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day that treatment commenced and then at weekly intervals for the duration of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
-- Food intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at start and prior to the completion of the 13 week treatment
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, Halothane anaesthesia
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: Haemoglobin (Hb), Red cell count (RBC), Platelets (PLT), Reticulocytes (RET), White cell count (WBC), Lymphocytes (LYM), Neutrophils (NEU), Monocytes (MON), Eosinophils (EOS), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Several indices are derived from these measurements: MCV - mean cell volume (mean of red blood cell volume histogram - femtolitres), HCT - haematocrit (red blood cell count times the MCV - litre/litre), MCH - mean cell haemoglobIn (haemoglobin divided by the red blood cell count picograms), MCHC - mean cell haemoglobin concentration (haemoglobin divided by the HCT grams/100 ml red cells), the following blood coagulation factors were measured: Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: in plasma: Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Aspartate transaminase, Alanine transaminase, Lactate dehydrogenase, 1-Hydroxybutyrate dehydrogenase, Creatine kinase, Alkaline phosphatase (pH 9.8), Pseudocholinesterase, 5-Nucleotidase, Triglyceride, Total cholesterol, Urea, Glucose, Creatinine; in serum: Total protein, Albumin; Serum electrophoresis separation yas performed on cellulose acetate plates followed by staining with Ponceau S and quantification of the fractions using the Profil Ecran scanning densitometer were performed for: Albumin Alpha-1-globulin, Alpha-2-globulin, Beta globulin, Gamma globulin, Albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from a selection of the 13 week rats (eight male and eight female rats per group) and the recovery rats prior to treatment, after 32 and 88 days of treatment, and after 13 weeks treatment plus 32 days recovery time for recovery rats only.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined analytically: Urine volume, Refractive index, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Urea, Creatinine N-acetyl-beta-D-glucosaminidase, Gamma glutamyl transferase, Alanine aminopeptidase
- Parameters examined semi-quantitative using dip-stick strips: leukocytes, nitrite, glucose, protein, erythrocytes and haemoglobin

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
- All the animals which survived to the completion of the 13 weeks treatment feeding period and at the end of the recovery period received a detailed
necropsy. All macroscopic abnormalities were recorded as well as an assessment of the level of intra-abdominal fat deposition.
- Brain, heart, liver, kidneys, spleen, testes, caecum, empty caecum and adrenal glands were weighed prior to fixation. The ratio of organ weight to 100 g of body weight at necropsy was calculated in each case and recorded as relative organ weight.
-- The following tissues were taken from each rat and preserved in 10% buffered formalin: Adrenal glands, Brain, Colon, Heart, Kidneys, Lungs, Mammary glands, Ovaries and fallopian tubes, Pituitary, Sciatic nerve, Sternum, Thyroid and parathyroid, Uterus, Aorta, Caecum, Duodenum, Ileum, Larynx Lymph nodes (cervical and mesenteric), Muscle, Prostate, Spinal cord, Stomach, Tongue, Bladder, Cervix, Head, Jejunum, Liver, Oesophagus, Pancreas, Rectum, Spleen, Thymus, Trachea
-- The following tissues were taken from each rat and preserved in Bouin's fixative: Epididymides, Seminal vesicles, Vagina, Femur and stifle joint, Skin, Sali vary glands, Testes
-- The eyes and harderian glands were taken from each rat and fixed in Davidson's fluid.

HISTOPATHOLOGY: Yes
- Tissues were processed by conventional histological methods into paraffin wax and sections 4µm (nominal) in thickness were prepared and stained with haematoxylin and eosin for histological examination. Additional portions of liver were fixed in 10% formol saline for the preparation of cryostat sections that were subsequently stained with Oil Red 0 to demonstrate neutral fat.
- Full histological examination was confined to rats from the high dose group (1.00% test item) and control group.

Postmortem examinations (offspring):

not applicable

Statistics:

- For body weights, food intakes, food efficiencies and water intakes, haematology, clinical chemistry and organ weights (including the ratio of organ weight to body weight at terminal kill), a statistical analysis was conducted using Student's t-test, F-test and Duncan's Multiple range test.
- A t-test versus control was used to show any significant differences between control group and any of the other treatment levels at the 5%, 1% and 0.1% probability levels. In conjunction with the F-ratio test in the analysis of variance, a Duncan's multiple range test at the 5% protection level was included to allow all pair-wise comparisons amongst the treatment levels to be carried out.

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

no effects observed

Description (incidence and severity):

no toxic symptoms, signs of distress or decrease in activity

Mortality:

no mortality observed

Description (incidence):

All rats survived to the end of the 13 week feeding period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Statistically significant lower body weights and body weight gains were observed over the 13 weeks of treatment for only the female rats fed 0.10%
test item when compared with controls. These were both considered to be marginal effects and were not dose related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- No statistically significant differences in food intakes were observed over the 13 weeks of treatment for either male or female rats fed the test item when compared with controls.
- the dietary dose levels compared to the following mean daily ingestion levels based on product and based on the a.i. of 33.8 %
-- 0.04 %: 28 mg test item/kg body weight/day; 9.5 mg a.i./kg bw/day
-- 0.10 %: 71 mg test item/kg body weight/day; 24 mg a.i./kg bw/day
-- 0.40 %: 288 mg test item/kg body weight/day; 97 mg a.i./kg bw/day
-- 1.00 %: 731 mg test item/kg body weight/day; 247 mg a.i./kg bw/day

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Main study groups:
- There was no evidence of an adverse effect of treatment in any of the tissues examined including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.
.
-- Caecum: There was no evidence of an effect of treatment on the morphology of the caecum.
-- Liver: There were no findings to explain decreased liver weight in the rats fed 1.00% test item.
-- Incidental findings: A variety of incidental findings was recorded in rats from all groups with no evidence of an effect of treatment on their character, incidence or distribution. The findings are consistent with the normal spectrum of spontaneous lesions encountered in young laboratory rats.
Recovery groups:
- Since there was no evidence of an adverse effect of treatment in any of the tissues examined, no histological evaluation was undertaken for the recovery rats.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Further details are given in the IUCLID section "Repeated dose toxicity".

Key result

Dose descriptor:

NOEL

Effect level:

247 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The results from the evaluation of reproductive organs, especially organ weights of ovary and testis and histopathology of gonads from this 90 day rat feeding study with 38 day recovery revealed no indications of any substance-related effects up to and including the highest test dose of 1% in diet, corresponding to 247 mg a.i./kg bw/day.

Executive summary:

In a subchronic toxicity study according OECD guideline 408, Coco AAPB (a.i. 33.8%) was administered to 12 male and 12 female Colworth Wistar rats per dose via food at dose levels of 0.00%, 0.04%, 0.10%, 0.40% and 1.00% (corresponding to 0, 28, 71, 288 and 731 mg product/kg bw (9.5, 24, 97 and 247 mg a.i./kg bw/day)) for 90 days. Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 32 days without treatment to detect recovery from, or persistence of, toxic effects. Concentrations in diet formulations were analytically verified and substance intake was calculated from recorded food consumption.

The substance was tolerated without any systemic effects relevant in view of an potential serious health risk for humans. Up to and including the highest dose tested of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day), there were no dose related effects on mortality, clinical signs, body weight, food consumption, water consumption, haematology, urinalysis and histopathology including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.

The only treatment related effects ascertainable at termination of treatment but not after the recovery period were reduced food conversion efficiencies and organ weight changes in the caecum and liver. The enlarged caecum observed at necropsy in some male and female rats fed 0.40% and 1.00% Coco AAPB was reflected in the statistically significant increases recorded in the absolute and relative, full and empty caecal weights for both male and female rats fed 1.00% Coco AAPB. These animals fed 1.00% Coco AAPB also showed reduced absolute and relative liver weights, reduced abdominal fat depots corresponding to the reduced food efficiency and several possibly associated plasma and serum biochemical changes. There was no histopathological correlate for the organ weight changes in caecum and liver. All alterations were completely reversible in the recovery group after 32 days without treatment.

Enlargement of the rat caecum with or without subsequent effects on caecum weight, food conversion and nutritional status is a common and frequently response to feeding poorly-absorbable or osmotically-active substances, such as xylitol, sorbitol, sucralose or natural sugars like d-ribose, general changes in nutritional diet composition or application of compounds with effects on the caecal microflora. In the absence of histopathological changes, the rat caecum alterations are taken as physiological adaptive responses and considered to be of no toxicological significance. An increase in liver weight without any histopathological correlate is commonly not considered to reflect an adverse effect but should be considered as an adaptive metabolic response which in known to be reversible. As also in this study, the increase in liver weight was without any histopathological correlate and has been proved to be reversible, the liver weight alteration is not considered to be an adverse effect relevant in view of an potential serious health risk for humans.

Therefore, the NOEL derived from this study relevant in view of a potential serious health risk for humans is the highest tested dose of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day).

Reference 3

Endpoint:

fertility, other

Remarks:

assessment of reproduction organs during Repeated Dose 90-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) (May 12, 1981)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CF (SD)BR Sprague Dawley
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: males: 115 - 174 g, females: 97 - 150 g
- Fasting period before study: no
- Housing: individually housing in solid floor macrolone cages of type III with stainless steel lids
- Diet: ad libitum, commercial powdered diet for laboratory animals (Ssniff R 10, Ssniff Spezialdiäten GmbH, 4770 Soest, West Germany); one exception of an overnight fast prior to blood sampling
- Water: ad libitum, tap water
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25°C (except on 13 occasions when temperature was once below 19°C and 6 x above 25 °C)
- Humidity: 30 - 70 %
- Photoperiod: artificial (fluorescent) light, 12 hrs dark / 12 hrs light)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The aqueous test item was further diluted with aqua destillata to achieve the scheduled doses.

VEHICLE
- Justification for use and choice of vehicle (if other than water): used vehicle was water
- Concentration in vehicle:
-- nominal: at dose levels of 0, 250, 500, and 1000 mg/kg bw/day: week 1 and 2: 0 g/500 mL, 12.5 g/500 mL, 25 g/500 mL, 50 g/500 mL
week 13: 0 g/800 mL, 20 g/800 mL, 40 g/800 mL, 80 g/800 mL
-- analytical measured (median values of four/two measurements each): at dose levels of 0, 250, 500, and 1000 mg/kg bw/day: week 1 and 2: 0 g/500 mL, 14.7 g/500 mL, 24 g/500 mL, 59.95 g/mL; week 13: 0 g/800 mL, 23 g/800 mL, 39.25 g/800 mL, 87.2 g/800 mL
- Total volume applied: 10 mL/kg bw

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (10 mL each for testing laboratory as reference sample and 10 mL for the study sponsor) of each test formulation were taken freshly and under conditions of use (first day and after seven days) in weeks 1, 2, and 13 and were sent to the study sponsor for analysis. The test article concentrations in the test article mixtures as well as the stability over seven days resulted to be good.

Duration of treatment / exposure:

Duration of test: 92 days
Exposure period: 90 days

Frequency of treatment:

5 days/week

Details on study schedule:

animals were not mated

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

based on product, corresponding to 75 mg a.i./kg bw/d

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

based on product, corresponding to 150 mg a.i./kg bw/d

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

based on product, corresponding to 300 mg a.i./kg bw/d

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The selection of the dose levels was based on the results of a preliminary toxicity study in the rat (HLD Project No. 348-156), see study 61789-40-0_8.6.1_Goldschmidt_1991_OECD 408_14-d dose finding also cited in this IUCLID

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes; All animals were examined twice daily at the beginning and end of the working day for morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Observation of ill health or overt signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during the treatment period, and on the day of necropsy or the day before

FOOD CONSUMPTION: yes
- Food consumption of the males and females was recorded twice weekly during the treatment period
- Food consumption was calculated as mean daily food consumption per measuring period

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals of group 1 (0 mg/kg bw/d) and 4 (1000 mg/kg bw/d) were examined once predose and during final week of treatment. Group 2 (250 mg/kg bw/d) and 3 (500 mg/kg bw/d) were additionally examined during the final week of treatment
- Dose groups that were examined: all dose groups
- Parameters examined: ocular fundus with macula lutea, papilla, and ocular vessels; before the examination, mydriatic agent (Mydriaticum "Roche"@,Hoffmann-La Roche Aktiengesellschaft, 7889 Grenzach-Wyhlen 1, Germany) was instilled into the eyes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during the final week of treatment (week 13)
- Anaesthetic used for blood collection: Yes; Blood samples were collected by orbital sinus puncture under light ether anesthesia
(Diethyl ether, Asid Bonz & Sohn GmbH, 7030 Böblingen, Hoechst Aktiengesellschaft, Germany)
- Animals fasted: Yes, overnight fast (about sixteen hours)
- How many animals: all animals
- Parameters examined: hemoglobin concentration (Hb), mean cell volume (MCV), red blood cell count (RBC) and derived indices: mean cell hemoglobin (MCH),
packed cell volume (PCV), and mean cell hemoglobin concentration (MCHC), prothrombin time, total and differential white blood cell count (WBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during the final week of treatment (week 13)
- Animals fasted: Yes, overnight fast (about sixteen hours)
- How many animals: all animals
- Parameters examined: glutamate oxalacetate transaminase (GOT, AST), glutamate pyruvate transaminase (GPT, ALT), alkaline phosphatase (AP), sodium (Na+), potassium (K+), chloride (Cl-), total protein (TP), blood urea (BU), albumin, albumin/globulin ratio (A/G ratio), glucose, cholesterol, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: during the final week of treatment (week 13)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, Urine samples were collected by placing the animals in metabolism cages at 14.00 hours or 15.00 hours without access to food or water. All urine passed between 14.00 hours and 06.00 hours or between 15.00 hours and 07.00 hours the following morning, respectively, were then collected.
- Parameters examined: measured: ph, volume, specific gravity; semiquantitative estimations of protein, blood, glucose, ketones, bilirubin, urobilinogen, reducing substances, colour, microscopy of centrifuged deposits

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, A full external and internal examination was made and all lesions were recorded. The necropsies were conducted over three days. As far as possible, equal numbers of male and female animals were killed on each day.
- Parameter examined: Individual organ weights; organ / body weight ratio (%), individual animal pathology
- The following organs were weighed before fixation: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroids (with para-thyroids), paired organs were weighed separately.

HISTOPATHOLOGY: Yes
- Samples of the following tissues (with exception of the eyes which were fixed in Davidson's fluid) were preserved in 10% neutral buffered formalin: adrenals, aorta (arch and anterior abdominal), bone marrow, brain (cerebral, cortex, thalamus, midbrain, medulla, cerebellum), cecum, colon, duodenum, epididymides, esophagus, eyes (and optic nerve), heart, ileum, jejunum, kidneys, liver, lungs (with mainstem bronchi), lymph nodes (mandibular and mesenteric), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submaxillary), sciatic nerve, seminal vesicle, skeletal muscle, skin and mammary gland, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids (with parathyroids), tongue, trachea, urinary bladder, uterus, all unusual lesions (according to OECD guideline 408)
- Samples examined: The above tissues from all animals in groups 1 and 4 were embedded in paraffin wax, sectioned at a nominal thickness of 5 µm, stained with hematoxylin and eosin, and were examined by the study pathologist. Due to treatment-related histopathological changes seen in high dose animals, stomach tissue of group 2 and 3 animals had been additionally examined microscopically.

Postmortem examinations (offspring):

not applicable

Statistics:

For body weight, body weight gain, and food consumption, the Levene's test for homogeneity of variance was performed followed by one-way Analysis of Variance.
In case of significant results for the ANOVA (p < 0.01 and p < 0.05), the Dunnett's test for multiple group comparisons (p < 0.05) was performed.
For organ weights, the Analysis of Variance was performed with one factor TREATMENT followed by the Student-Newman-Keuls test.
For clinical chemistry data, hematology data, and organ/body weight ratio, the Analysis of Variance was performed with one factor TREATMENT - based on taking the ranks of the variables - and followed by the Student-Newman-Keuls test for multiple group comparisons.
The statistical evaluation was performed with the standard software package SAS (Statistical Analysis System) release 6.03 excluding the analysis for body weight, body weight gain, and food consumption which was performed with the statistical package of the on-line data collection system TERASYS.

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no clinical changes observed in any animal throughout the experimental period which would be related to treatment with the test article. On a few occasions, wounds, cuts, and scratches, nose bleed, crusted nose, and nasal discharge were observed in single animals. The findings are considered to be not treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- males: One male control animal and one group 3 (intermediate dose group) male died accidentally on days 29 and 19 respectively. These unscheduled mortalities were considered to be incidental and due to experimental procedure.
- female: One female group 3 animal (intermediate dose group) died accidentally on day 26 and three high dose females died accidentally on days 26, 41, and 90 of study. These unscheduled mortalities were considered to be incidental and due to experimental procedures.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Body weight gain (day 1 to 91) was considered normal for male and female animals of all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There was no treatment-related adverse effect on overall food consumption (weeks 1 to 13) for test item treated males and females.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Forestomach: Forestomach gastritis was seen in high and mid dose males and females. From the samples examined the foresomach gastritis was chronic and diffuse in affected animals and was characterised by squamous hyperplasia, with submucosal oedema and inflammatory cell infiltration in some animals. The severity of the forestomach gastritis was judged by the pathologist as minimal to moderate.
-- high dose group: Forestomach gastritis was present in six high dose group males, and three high dose females.
-- mid dose group: Forestomach gastritis was present in two intermediate dose group males and two intermediate dose group females.
-- low dose group: Forestomach gastritis was not present in the stomachs of low dose group animals.
- All other examined organs including epididymides, testes, prostate, seminal vesicle, ovaries, mammary gland and uterus: There was no evidence of any systemic toxicity due to test article administration in any of the other organs examined.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Further details are given in the IUCLID section "Repeated dose toxicity".

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There were no adverse test substance-related effects on reproductive organs (organ weights of ovary and testis and histopathology of gonads) up to the highest tested dose level.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no adverse test substance-related effects on reproductive organs (organ weights of ovary and testis and histopathology of gonads) up to the highest tested dose level.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The results from the evaluation of reproductive organs, especially organ weights of ovary and testis and histopathology of gonads from this 90 day rat gavage study revealed no indications of any substance-related effects up to and including the highest test dose of 300 mg a.i./kg bw/d ( = 1000 mg product (30.3% a.i.)/kg bw/d).

Executive summary:

In a subchronic toxicity study according OECD guideline 408 (1981), C8 -18 AAPB (30.3% a.i.) was administered to 10 male and 10 female Sprague-Dawley rats per dose by gavage at dose levels of 0, 250, 500, 1000 mg/kg bw/day (corresponding to ca. 75, 150, and 300 mg active ingredient/kg bw) for 90 days. The aqueous test item was further diluted with aqua destillata to achieve the scheduled doses. Concentrations in test formulations were analytically verified. The substance was tolerated without any systemic effects.

Up to and including the highest dose tested of 1000 mg/kg bw, there were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights including weights of ovaries and testes, systemic organ pathology and histopathology including inspection of epididymides, testes, prostate, seminal vesicle, ovaries, mammary gland and uterus.The only treatment related effect seen in this study was a local inflammatory response at the site of application (forestomach gastritis) most probably caused by an irritant effect of the test item. These appeared in gross pathology findings in form of some stomach ulcer at fundus and cardia region in one male and one female rat at 1000 mg/kg bw/day, and in microscopic findings in form of squamous hyperplasia, submucosal edema, inflammatory cell filtration at a dose level of >= 500 mg/kg bw/day (2/10 male and 2/10 female rats at a dose level of 500 mg/kg bw, and at 1000 mg/kg bw in 6/10 males and 3/10 females).

Therefore, the NOEL derived from this study relevant to human DNEL calculation is the NOEL for systemic effects which is the highest tested dose of 300 mg a.i./kg bw/d ( = 1000 mg product (a.i. ca. 30%)/kg bw/d.

The LOEL local effects (500 mg/kg bw/day, corresponding to ca. 150 mg active ingredient/kg bw), based on local irritative effects at the site of application (forestomach gastritis), is judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans.

Reference 4

Endpoint:

fertility, other

Remarks:

90 d repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar strain, Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean. (males: 156-176 grams; females: 93-123 grams)
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 23.4
- Humidity (%): 41 - 95
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 17 October 2011 - 19 January 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for density and purity of the test substance.

VEHICLE:
Water

DOSE VOLUME:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight

Details on mating procedure:

no mating

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted during the treatment phase, according to a validated method (NOTOX project 454117). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations; in weeks 1, 6 and 13). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use in Week 6. This was considered to have been introduced during sample pretreatment, since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

At least 90 days.

Frequency of treatment:

Once daily, 7 d/w.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000
Basis:
actual ingested

No. of animals per sex per dose:

10

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with the test item by daily gavage in the rat (NOTOX project 497623).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase pre-dose clinical observations were performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs was recorded.
Arena observations were conducted prior to dosing instead of after dosing. No peak effect of occurrence of clinical signs was noted during the dose range finding study. Therefore, the timing of conduct of the arena observations was considered not critical.

BODY WEIGHT: yes
- Time schedule for examinations: Weekly.

FOOD EFFICIENCY: yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data
Food consumption was measured off-line on Day 9 instead of Day 8. Based on conducted measurements, an adequate assessment of the food intake could be made. A correction was made afterwards in the calculated food intake data to account for the difference in measurement period of food intake during the first two weeks.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes
- Time schedule for examinations: at pretest: all animals (including spare animals), at week 13: Groups 1 and 4.

HAEMATOLOGY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12-13 of treatment
- Dose groups that were examined: all animals of Groups 1 and 4
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: yes
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS: yes
Organs checked according to test guidelines

HISTOPATHOLOGY: yes
According to test guidelines
For one animal of Group 4 one mandibular lymph node was available for histopathology. Sufficient data was available for histopathological evaluation.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabs, a broken tail apex, elongated teeth (indicated in the tables as “broken upper incisors”) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD EFFICIENCY
Food consumption before or after allowance for body weight was similar between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest or in week 13 consisted of pinpoint or multifocal corneal opacities, central lens opacity, persistent hyaloid vessel remnants, posterior cataract due to hyaloid remnants, and focal corneal oedema. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain, and therefore considered to be of no toxicological relevance.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower white blood cell (WBC) red blood cell counts in males at 1000 mg/kg, higher mean corpuscular haemoglobin (MCH) in males and females at 1000 mg/kg, higher mean corpuscular volume (MCV) in males at 100 and 300 mg/kg, lower reticulocyte counts in females at 300 mg/kg, lower haematocrit level in females at 100 mg/kg, higher mean corpuscular haemoglobin concentration (MCHC) in females at 100, 300 and 1000 mg/kg, and higher platelet counts in females at 300 and 1000 mg/kg.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower sodium and chloride levels in males at 100 mg/kg, higher total bilirubin and urea levels in females at 1000 mg/kg, higher creatinine levels in females at 100, 300 and 1000 mg/kg, and lower potassium levels in females at 100 mg/kg.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals of the control group and at 1000 mg/kg. Motor activity was similar between the 1000 mg/kg and control group. Both groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. Statistically significant changes in organ weights were considered not to be a sign of toxicity as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower absolute and relative spleen and prostate weight in males at 100 mg/kg, lower absolute and relative seminal vesicle weight in males at 100 and 300 mg/kg, higher absolute brain weight in females at 300 mg/kg, and lower absolute and relative spleen weight in female at 300 mg/kg.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included red foci on the lungs, an accessory liver on the caudate lobe, pelvic dilation of the kidneys, flaccid seminal vesicles, a tan focus on the preputial or clitoral glands, tan discolouration of the clitoral glands, enlarged spleen with irregular surface, a bent tail bone, a yellowish hard or soft nodule on the epididymal adipose tissue, fluid in the uterus, red discolouration of the mandibular lymph nodes and alopecia.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects on reproductive organs up to the limit dose

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/d was established.

Executive summary:

In a subchronic toxicity study (according to OECD Guideline 408), the testsubstance was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg for 90 consecutive days.

There were no compound related effects in mortality, clinical signs, functional observations, ophthalmoscopy, body weight and weight gain, food consumption and food efficiency, hematology, clinical chemistry, organ weights, gross and histologic pathology or neurobehaviour.

Especially no effects on the weights on reproductive organs (testes, epididymides, prostate, seminal vesicles including coagulating glands,

uterus, ovaries) were noted. Histopathological examination revealed no microscopic findings in the reproductive organs

(testes, epididymides, prostate, seminal vesicles, uterus, ovaries, cervix, vagina).

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/d was established.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements for a OECD 408 study in rats.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

All available data are reliable and of high quality (guideline studies, GLP).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable data from a 28 d repeated dose toxicity study in rats with additional focus on reproductive organs is available for C8-10 Alkylamidopropyl betaine. Further repeated dose toxicity studies are available for the closely related source substances C8-18 AAPB, C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine. A justification for read-across is given below.

 

In a subacute toxicity study according to OECD guideline 407 (2008) and EU method B.7 (2008) C8-10 Alkylamidopropyl betaine (34.65% a.i.) was administered to 5Hsd: Sprague Dawley SD rats/sex/dose in purified water by gavage at dose levels of 0 (control), 100, 300 and 500 mg a.i./kg bw/day for 28 consecutive days. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Neurotoxicity assessment did not reveal any treatment-related effects. No changes on body weight and food consumption were noted.

The lymphocytosis and monocytosis seen in single females dosed at 300 and/or 500 mg/kg bw/day showed reversibility at the end of the recovery period or comparability to control data. No toxicological relevant effects in coagulation and clinical chemistry parameters were observed. No differences were reported in terminal body weights and organ weights between treated and control animals and no treatment-related changes were noted at macroscopic and microscopic observations.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

On the basis of the results obtained in this study, the dose level of 500 mg a.i./kg bw/day was considered the NOAEL.

 

In the 90-day repeated dose toxicity studies (tested doses up to and including approx. 300 mg a.i./kg bw/d) in rats conducted with C8-18 AAPB or C8-18 and C18 unsatd. AAPB, there were no histopathological changes in reproductive organs (seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix, vagina) and no effects on reproductive organs weights (testes, ovaries). In the prenatal developmental toxicity study C8-18 AAPB showed no teratogenic activity, and embryotoxic effects were found only at the maternal toxic dose level. Taking into account the overall low toxic activity of the substances, particularly with regard to the missing adverse effects on reproductive organs or tissues in the repeated dose toxicity studies as well as in the developmental toxicity study, the missing teratogenic activity, the fact that embryotoxic effects were found only at the maternal toxic dose level and the toxicodynamics of both substances, which is primarily based on its irritancy, fertility-specific effects are highly unlikely to occur. Therefore, further reproductive toxicity studies do not need to be conducted.

 

In a subchronic toxicity study (according to OECD Guideline 408), Formamidopropylbetaine was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg for 90 consecutive days.

There were no compound related effects in mortality, clinical signs, functional observations, ophthalmoscopy, body weight and weight gain, food consumption and food efficiency, hematology, clinical chemistry, organ weights, gross and histologic pathology or neurobehaviour.

Especially no effects on the weights on reproductive organs (testes, epididymides, prostate, seminal vesicles including coagulating glands, uterus, ovaries) were noted. Histopathological examination revealed no microscopic findings in the reproductive organs (testes, epididymides, prostate, seminal vesicles, uterus, ovaries, cervix, vagina).

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/d was established.

 

In accordance with Annex IX column 2 of the REACH Regulation (EC) No 1907/2006, the performance of an Extended One-Generation Reproductive Toxicity Study is not required. The substance is of low systemic toxicity as indicated by 28 d NOAEL of 500 mg/kg bw/d, which was the highest tested dose level. No indication of any systemic toxicity relevant in view of a potential health risk for humans was found in the sub-chronic studies conducted with closely related source substances, including reproductive organs. From developmental toxicity data obtained with with closely related source substances, there is no evidence for teratogenic effects. The substance does not have genotoxic properties as proven in the full data set including in vivo data on the substance itself or closely related source substances.

The use profile of the substance indicates that relevant exposure to humans occurs via the dermal route. Reliable, relevant and adequate toxicokinetic data from an in vitro study on human skin conducted with a closely related source substance showed a dermal resorption rate of 0 %.

Based on the above specified toxicological and toxicokinetic data, it can be proven that the substance is of low toxicological activity and that no systemic absorption occurs via the relevant route of exposure. Therefore, further reproductive toxicity studies do not need to be conducted.

Further, in accordance with Annex XI, section 1.2 of the REACH Regulation (EC) No 1907/2006, the performance of an Extended One-Generation Reproductive Toxicity Study is scientifically unjustified. As indicated above there is no indication of any systemic toxicity of the substance relevant in view of a potential health risk for humans, neither from sub-chronic data nor from developmental toxicity data.

In conclusion, further testing on vertebrate animals in an Extended One-Generation Reproductive Toxicity Study is unjustified for animal welfare reasons.

There are no data gaps for effects on fertility. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.

 

Conclusion

The NOEL for effects on fertility is derived from the 90 d repeated dose toxicity study with additional focus on reproductive organs, which was conducted with C8-18 AAPB which is the highest tested dose of 300 mg a.i./kg bw/day.

 

There are no data gaps for the endpoint fertility. No human data are available. However, there is no reason to believe that these results from rabbit would not be applicable to humans.

 

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

 

This read-across approach is justified based on structural similarities. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a variety in their carbon chain length, which obviously does not have a relevant impact on toxicity toxicity to reproduction as demonstrated by the available data on the target and source substances.

 

a. Structural similarity and functional groups

The target substance C8-10 Alkylamidopropyl betaine is a UVCB substance manufactured from fatty acids (C8 and C10) and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

The source substance C8 -18 AAPB is a UVCB substance manufactured from natural fatty acids or oils with N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance C8-18 and C18 unsatd. AAPB is a UVCB substance manufactured from natural fatty acids or oils with N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance Formamidopropylbetaine is a monoconstituent substance manufactured from formic acid and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

 

b. Differences

Differences in chemical and other intrinsic properties of the target and source substances could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length lead to a rising average lipophilicity as can be seen from the increasing log Kow from Formamidopropylbetaine (log Kow: -3.3), C8-10 Alkylamidopropyl betaine (log Kow: 2.2), C12 AAPB (log Kow: 3.54), C8-18 AAPB (log Kow: 4.23).

However, based on the available toxicological data it is demonstrated, that this read-across approach is nevertheless reliable.

 

- Different amounts of unsaturated fatty ester moieties:

The source substance C8-18 and C18 unsatd. AAPB contains considerable amounts of unsaturated C18 chains, which represents a worst case with respect to some toxicological endpoints, mainly local effects (e.g. irritation, sensitisation). But in general, variability in the fatty acid moiety is not expected to be relevant to the intrinsic systemic or reproductive toxicity of the substances.

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of reproductive toxicity data

Endpoints	Source substances			Target substance
	C8-18 and C18 unsatd. AAPB	C8-18 AAPB	Formamidopropylbetaine	C8-10 Alkylamidopropyl betaine
Data from tepeated dose toxicity studies	sup_RA_Toxicity to reproduction: 61789-40-0_8.6.2_90days_Unilever_A03_FT890785   Key study   OECD TG 408, subchronic, rat, oral: feed   NOEL effects relevant to humans: 247 mg a.i./kg bw/d (highest tested dose, 1 % in feed, 731 mg/kg bw/d based on product (a.i. 33.8 %))   LOEL: 97 mg a.i./kg bw/day) (0.4% in feed, 288 mg/kg bw/d based on product (a.i. 33.8 %))   Reliability: 1 (reliable without restriction), GLP	sup_RA_Toxicity to reproduction: 97862-59-4_8.6.2_Goldschmidt_1991_OECD 408 key study OECD TG 408, subchronic, rat, oral: gavage NOEL systemic effects: 300 mg a.i./kg bw/d (highest tested dose, 1000 mg/kg bw/d based on product (a.i. ca. 30 %)) LOEL local effects: 150 mg a.i./kg bw/d (500 mg/kg bw/d based on product (a.i. ca. 30 %)) NOEL local effects: 75 mg a.i./kg bw/d (250 mg/kg bw/d based on product (a.i. ca. 30 %))   Reliability: 1 (reliable without restriction), GLP	sup_RA_Toxicity to reproduction: oral 90-day OECD 408 NOTOX 497622   OECD TG 408, subchronic, rat, oral: gavage   NOAEL = 1000 mg/kg bw/d No toxicologically relevant effects were observed up to the highest dose level tested.   Reliability: 1 (reliable without restrictions), GLP	sup_RA_Toxicity to reproduction: 73772-45-9 / 73772-46-0_8.6.2_Evonik_2016_OECD407   OECD TG 407, subacute, rat, oral: gavage   NOAEL = 500 mg/kg bw/d No toxicologically relevant effects were observed up to the highest dose level tested   Reliability: 1 (reliable without restrictions), GLP
Prenatal developmental toxicity	No data	WoE_Developmental toxicity / teratogenicity: 97862-59-4_8.7.2_CESIO_2004_OECD 414 Key study OECD TG 414, rat, oral: gavage NOEL maternal toxicity: 100 mg a.i./kg bw/day NOEL embryotoxicity: 300 mg a.i./kg bw/day NOEL teratogenicity: 1000 mg a.i./kg bw/day Maternal toxic effects: yes Embryotoxic / teratogenic effects: yes   Reliability: 1 (reliable without restriction), GLP	WoE_RA_Developmental toxicity / teratogenicity NOTOX 497620   OECD TG 414, rat, oral: gavage   NOEL maternal toxicity: 1000 mg a.i./kg bw/day NOEL teratogenicity: 1000 mg a.i./kg bw/day   Maternal toxic effects: no Embryotoxic / teratogenic effects: no   Reliability: 1 (reliable without restrictions), GLP	No data, read-across

 

In the repeated dose toxicity studies performed according to the corresponding OECD Guidelines on the target substance itself as well as on the source substances C8-18 AAPB, C8-18 and C18 unsatd AAPB and Formamidopropylbetaine, up to and including the respective highest tested dose levels, no indication of any effects of the substances to reproductive organs were observed.

In the prenatal developmental toxicity study (tested doses up to and including 1000 mg a.i./kg bw/d) C8-18 AAPB showed no teratogenic activity, and embryotoxic effects were found only at the maternal toxic dose level. The second source substance Formamidopropylbetaine showed no teratogenic or embryotoxic activity.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

Repeated dose toxicity studies:

The studies were conducted according to OECD Guideline 407 or 408 and were reliable without restrictions (RL1, GLP).

Prenatal developmental toxicity studies:

The studies were conducted according to OECD Guideline 414 and reliable without restrictions (RL1, GLP).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

Based on structural similarities of the target and source substances as presented above and in more detail in the general justification for read across as well as on similarly low systemic toxicity shown in the available repeated dose toxicity studies, it can be concluded that the available data from the source substances C8-18 AAPB, C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine are also valid for the target substance C8-10 Alkylamidopropyl betaine.

In the subacute repeated dose toxicity study in rats conducted with the target substance C8-10 Alkylamidopropyl betaine, there were no histopathological changes in reproductive organs (epididymides, seminal vesicles, prostate + coagulating glands, testes, ovaries + oviduct, uterus, cervix, vagina) and no effects on reproductive organs weights (epididymides, testes, prostate, ovaries). Spermatogenic staging and oestrous cycle were normal.

In the subchronic repeated dose toxicity studies in rats conducted with C8-18 and C18 unsatd. AAPB and C8-18 AAPB, there were no histopathological changes in reproductive organs (seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix, vagina) and no effects on reproductive organs weights (testes, ovaries).

In the subchronic toxicity study in rat conducted with the source substance Formamidopropylbetaine no effects on the weights on reproductive organs (testes, epididymides, prostate, seminal vesicles including coagulating glands, uterus, ovaries) were noted. Histopathological examination revealed no microscopic findings in the reproductive organs (testes, epididymides, prostate, seminal vesicles, uterus, ovaries, cervix, vagina).

Taking into account the overall low toxic activity of the substances, particularly with regard to the missing adverse effects on reproductive organs or tissues in the 28 day and 90 day studies as well as in the developmental toxicity studies, the missing teratogenic activity, the fact that embryotoxic effects – if at all – were found only at the maternal toxic dose level, fertility-specific effects are highly unlikely. 

Effects on developmental toxicity

Description of key information

- Prenatal developmental toxicity study; oral (gavage); rat (CD/Crl:CD, 25/group, dosed from day 5 through 19 post coitum); OECD Guideline 414; GLP; RL1; NOEL(maternal toxicity) = 100 mg/kg bw/d / NOEL(embryotoxicity) = 300 mg/kg bw/d / NOEL(teratogenicity) = 1000 mg/kg bw/d, read-across: C8-18 AAPB.
- Prenatal developmental toxicity study; oral (gavage); rat (Wistar, 22/group, dosed from day 6 through 19 post coitum); OECD Guideline 414; GLP; RL1; NOEL(maternal toxicity, embryotoxicity, teratogenicity) = 1000 mg/kg bw/d, read-across: Formamidopropylbetaine

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 December 2011 - 26 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: males and females. Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight of females range at start of post-coitum was 215 gram.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-coitum: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days
- Identification: Animals were only identified by tattoo on the foot and not by earmark as well. Animals were individually housed with unique identification, thus this has no impact on the study’s integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 21.5°C
- Humidity (%): 42 - 89%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 20 December 2011 - 26 January 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Corrections were made
for the purity and the density of the test substance.

Storage conditions of formulations:
At ambient temperature.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (06 January 2012), according to a validated method (NOTOX Project 454117). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and no test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male ).
- Age at mating of the mated females in the study: Approximately 12 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Duration of treatment / exposure:

Females were exposed from Days 6 to 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Duration of treatment: From Days 6 to 19 post-coitum, inclusive.

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of the pilot study (NOTOX Project 497618 (see attached document), based on the results from the 14-day pilot to the 90-day study (NOTOX Project 497923) and the results of the 90-day study (NOTOX Project 497622) available at initiation of the
main prenatal developmental toxicity study.
- Rationale for animal assignment: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from Day 0 post-coitum onwards. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

NEUROBEHAVIOURAL EXAMINATION:
No

BODY WEIGHT:
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum

FOOD CONSUMPTION:
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION :
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: first 10 pregnant females/group. Instead of 10 animals, blood was taken from 14, 13, 12 and 13 animals from Groups 1, 2, 3, and 4, respectively. The additional data is reported and has no adverse impact on the study’s integrity.
- Parameters checked were: According to test guidelines


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: No the animals were not deprived of food overnight. Water was provided.
- How many animals: first 10 pregnant females/group. Instead of 10 animals, blood was taken from 13, 12, 12 and 13 animals from Groups 1, 2, 3, and 4, respectively. The additional data is reported and has no adverse impact on the study’s integrity.
- Parameters checked were: According to test guidelines

URINALYSIS:
No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses
- The number and distribution of embryo-fetal deaths
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
The uterus from one animal of Group 2 was not weighed Evaluation: Sufficient data is available for a thorough evaluation.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter of Groups 1 and 4
- Head examinations: Yes: half per litter

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss. Dead fetuses, early and late resporptions and pre- and post-implantation loss were compared using the litter as the statistical unit.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected / litter (%) = (number of viable fetuses affected / litter)/(number of viable fetuses/litter) x 100

Historical control data:

report contains historical control data on malformations and variations for the rat strain used. Number of studies evaluated: 23
Total No. Fetuses/Litters Examined Externally 4557 384
Total No. Fetuses/Litters Examined Viscerally 3740 384
Total No. Fetuses/Litters Examined Skeletally 3122 376

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were 2, 1, 2 and 2 non-pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the numbers of implantation sites, corpora lutea, pre-implantation or post-implantation loss up to 1000 mg/kg. Post-implantation loss was higher at 100 mg/kg, but in the absence of a dose response it was not considered treatment related.

Mortality: There were no unscheduled deaths in the study.

Clinical signs: There were no treatment related clinical signs noted up to 1000 mg/kg. Alopecia was an incidental clinical sign noted for control and treated animals, which had no association with treatment.

Body weights: Body weights and body weight gains remained unaffected by treatment up to 1000 mg/kg.

Food consumption: There were no treatment related effects on absolute or relative food consumption up to 1000 mg/kg.

Haematology: There were no toxicologically relevant effects on haematology parameters up to 1000 mg/kg. Significant reductions in red blood cell counts (all treated groups) and in haemoglobin and haematocrit values (both at 100 and 1000 mg/kg) were seen. However, these differences were likely due to relatively high means obtained for control animals that were attributable to high values for animal nos. 1 and 3 for all three parameters (high red blood cells for one female of the control group were also seen). In the absence of treatment related effects on any other parameter, these changes were not considered to be toxicologically relevant.

Clinical biochemistry: Clinical biochemistry parameters were unaffected up to 1000 mg/kg.

Macroscopic examination: There were no macroscopic findings attributable to treatment noted up to 1000 mg/kg. Incidental findings seen for control and treated animals included alopecia on the flanks, hind legs and/or abdominal regions. Tan discoloration of the thymus was also noted for one control female and one high dose female; this high dose female also had fluid in the uterus and cervix. These findings have no toxicological relevance.

Organ weights: There were no differences in uterine weights between control and treated animals up to 1000 mg/kg.

Maternal pregnancy data: There were 2, 1, 2 and 2 non-pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the numbers of implantation sites, corpora lutea, pre-implantation or post-implantation loss up to 1000 mg/kg. Post-implantation loss was higher at 100 mg/kg, but in the absence of a dose response it was not considered treatment related.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on the litter size for any group, nor were there any effects on the numbers of viable or dead fetuses.

Litter size: There was no effect of treatment on the litter size for any group, nor were there any effects on the numbers of viable or dead fetuses.

Sex ratio: The sex ratio was unaffected by treatment.

Fetal body weight: There were no treatment related effects on fetal body weights up to 1000 mg/kg.

External malformations and variations: There were no test substance-related effects on fetal external morphology. Two live fetuses and a dead fetus were observed with external malformations in the mouth region. One fetus at 100 mg/kg had a small lower jaw and one fetus at 1000 mg/kg had a cleft upper lip (bilateral), cleft palate (entire length) and small upper jaw. The cleft palate and small upper jaw of one fetus 1000 mg/kg were skeletally confirmed and in addition it appeared that this fetus had fused nasal bones and fused mandibles. The dead fetus at 100 mg/kg had craniorachischisis (the cranial vault and vertebral column were open through the sacral region) and a small upper jaw. No external developmental malformations were observed in any of the other fetuses and external variations were not observed in this study. Visceral malformations and variations: There were no test substance-related effects on fetal visceral morphology. Visceral malformations were observed 0(0), 3(2), 1(1) and 1(1) fetuses (litters) in the control, 100, 300 and 1000 mg/kg groups, respectively. At 100 mg/kg, the malformations observed were small eyes in one fetus (this fetus also had a small lower jaw at external examination), retroesophageal aortic arch in one fetus and internal hydrocephaly (observed at cephalic examination) in one fetus. At 1000 mg/kg, one fetus also had internal hydrocephaly and at 300 mg/kg, one fetus only had one testis and epididymis. In addition to these fetuses with visceral malformations, the dead fetus at 100 mg/kg had a malpositioned testis.
Visceral developmental variations observed in the test substance-treated groups were partially undescended thymus horns, small supernumerary liver lobes, liver appendix, dilated ureter, pale spleen, spot on stomach, supernumerary pancreas, discolored adrenal, right subclavian originating from the aortic arch and malpositioned left carotid.

Skeletal malformations and variations
There were no test substance-related effects on fetal skeletal morphology. Skeletal malformations were observed in 3(3) fetuses (litters) in both the control and 1000 mg/kg group. At 1000 mg/kg, two fetuses had a vertebral anomaly with associated rib anomaly. The anomaly in one fetus was located in the thoracic and lumbar vertebral column region and in one fetus in the cervical and thoracic region. Also at 1000 mg/kg, one fetus which was noted with a small upper jaw and cleft lip and cleft palate externally, had fused nasal bones and fused mandibles. The malformations observed in the control group were malpositioned metatarsal in one fetus, supernumerary metatarsal in one fetus and sternoschisis in one fetus. The mean litter incidence of ossified cervical centrum no. 1 was significantly decreased at 1000 mg/kg (23.2% per litter) compared to the control group value (36.0% per litter). Other skeletal developmental variations observed more than once in the test substance-treated groups were 14th rudimentary ribs, 7th cervical rudimentary ribs, unossified sternebra nos. 5 and 6, slightly to moderately malaligned sternebrae, reduced ossification of the skull, bent ribs, reduced ossification of vertebral centra, caudal shift of pelvic girdle and 14th full ribs.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

MATERNAL FINDINGS

Mortality: There were no unscheduled deaths in the study.

Clinical signs: There were no treatment related clinical signs noted up to 1000 mg/kg. Alopecia was an incidental clinical sign noted for control and treated animals, which had no association with treatment.

Body weights: Body weights and body weight gains remained unaffected by treatment up to 1000 mg/kg.

Food consumption: There were no treatment related effects on absolute or relative food consumption up to 1000 mg/kg.

Haematology: There were no toxicologically relevant effects on haematology parameters up to 1000 mg/kg. Significant reductions in red blood cell counts (all treated groups) and in haemoglobin and haematocrit values (both at 100 and 1000 mg/kg) were seen. However, these differences were likely due to relatively high means obtained for control animals that were attributable to high values for animal nos. 1 and 3 for all three parameters (high red blood cells for one female of the control group were also seen). In the absence of treatment related effects on any other parameter, these changes were not considered to be toxicologically relevant.

Clinical biochemistry: Clinical biochemistry parameters were unaffected up to 1000 mg/kg.

Macroscopic examination: There were no macroscopic findings attributable to treatment noted up to 1000 mg/kg. Incidental findings seen for control and treated animals included alopecia on the flanks, hind legs and/or abdominal regions. Tan discoloration of the thymus was also noted for one control female and one high dose female; this high dose female also had fluid in the uterus and cervix. These findings have no toxicological relevance.

Organ weights: There were no differences in uterine weights between control and treated animals up to 1000 mg/kg.

Maternal pregnancy data: There were 2, 1, 2 and 2 non-pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the numbers of implantation sites, corpora lutea, pre-implantation or post-implantation loss up to 1000 mg/kg. Post-implantation loss was higher at 100 mg/kg, but in the absence of a dose response it was not considered treatment related.

FETAL FINDINGS

Litter size: There was no effect of treatment on the litter size for any group, nor were there any effects on the numbers of viable or dead fetuses.

Sex ratio: The sex ratio was unaffected by treatment.

Fetal body weight: There were no treatment related effects on fetal body weights up to 1000 mg/kg.

External malformations and variations: There were no test substance-related effects on fetal external morphology. Two live fetuses and a dead fetus were observed with external malformations in the mouth region. One fetus at 100 mg/kg had a small lower jaw and one fetus at 1000 mg/kg had a cleft upper lip (bilateral), cleft palate (entire length) and small upper jaw. The cleft palate and small upper jaw of one fetus 1000 mg/kg were skeletally confirmed and in addition it appeared that this fetus had fused nasal bones and fused mandibles. The dead fetus at 100 mg/kg had craniorachischisis (the cranial vault and vertebral column were open through the sacral region) and a small upper jaw. No external developmental malformations were observed in any of the other fetuses and external variations were not observed in this study. Visceral malformations and variations: There were no test substance-related effects on fetal visceral morphology. Visceral malformations were observed 0(0), 3(2), 1(1) and 1(1) fetuses (litters) in the control, 100, 300 and 1000 mg/kg groups, respectively. At 100 mg/kg, the malformations observed were small eyes in one fetus (this fetus also had a small lower jaw at external examination), retroesophageal aortic arch in one fetus and internal hydrocephaly (observed at cephalic examination) in one fetus. At 1000 mg/kg, one fetus also had internal hydrocephaly and at 300 mg/kg, one fetus only had one testis and epididymis. In addition to these fetuses with visceral malformations, the dead fetus at 100 mg/kg had a malpositioned testis.

Visceral developmental variations observed in the test substance-treated groups were partially undescended thymus horns, small supernumerary liver lobes, liver appendix, dilated ureter, pale spleen, spot on stomach, supernumerary pancreas, discolored adrenal, right subclavian originating from the aortic arch and malpositioned left carotid.

Skeletal malformations and variations

There were no test substance-related effects on fetal skeletal morphology. Skeletal malformations were observed in 3(3) fetuses (litters) in both the control and 1000 mg/kg group. At 1000 mg/kg, two fetuses had a vertebral anomaly with associated rib anomaly. The anomaly in one fetus was located in the thoracic and lumbar vertebral column region and in one fetus in the cervical and thoracic region. Also at 1000 mg/kg, one fetus which was noted with a small upper jaw and cleft lip and cleft palate externally, had fused nasal bones and fused mandibles. The malformations observed in the control group were malpositioned metatarsal in one fetus, supernumerary metatarsal in one fetus and sternoschisis in one fetus. The mean litter incidence of ossified cervical centrum no. 1 was significantly decreased at 1000 mg/kg (23.2% per litter) compared to the control group value (36.0% per litter). Other skeletal developmental variations observed more than once in the test substance-treated groups were 14th rudimentary ribs, 7th cervical rudimentary ribs, unossified sternebra nos. 5 and 6, slightly to moderately malaligned sternebrae, reduced ossification of the skull, bent ribs, reduced ossification of vertebral centra, caudal shift of pelvic girdle and 14th full ribs.

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Effect Level (NOEL) for Formamidopropylbetaine was established as being at least 1000 mg/kg body weight/day.

Executive summary:

In a developmental toxicity study according to OECD guideline 414, the testsubstance was administered to 22 female Crl:WI(Han) rats/dose by gavage at dose levels of 0, 100, 3000 and 1000 mg/kg bw/day from days 6 to 19 post-coitum.

There were no maternal treatment-related effects in mortality, clinical signs, body weight, food consumption, or cesarean parameters observed in the 100, 300 and 1000 mg/kg/day groups. The maternal NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

There were no treatment-related effects in developmental parameters observed in the 100, 300 and 1000 mg/kg/day groups. The developmental NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rat.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-10-22 to 2004-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 22, 2001

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

Remarks:

certificate Stadt Hamburg, Germany

Limit test:

no

Species:

rat

Strain:

other: CD/Crl:CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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 iniectabilaia
- 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

Dose / conc.:

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

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

100 and 300 mg a.i./kg bw / day: no mortality; 1000 mg a.i. /kg bw / day: 1/20 on gestation day 15

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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
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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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 1000 mg a.i./kg bw / day: A severely and statistically significantly (at

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.
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
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 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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

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

Details on maternal toxic effects:

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.

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 - 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

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

number of resorptions (early, late and total) was increased in the high dose group (1000 mg/kg bw / d)

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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.

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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

External malformations:

no effects observed

Description (incidence and severity):

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 malformations:

no effects observed

Description (incidence and severity):

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 - 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 - 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.

Visceral malformations:

no effects observed

Description (incidence and severity):

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

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

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:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

All available data are reliable and of high quality (guideline studies, GLP).

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

No experimental data on prenatal developmental toxicity are available for the target substance  C8-10 Alkylamidopropyl betaine. However, reliable and relevant data are available for the closely related source substances C8-18 AAPB and Formamidopropylbetaine. A justification for read-across is given below.

 

In a prenatal developmental toxicity study performed according OECD guideline 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.

 

In a prenatal developmental toxicity study according to OECD guideline 414, Formamidopropylbetaine was administered to 22 female Crl:WI(Han) rats/dose by gavage at dose levels of 0, 100, 3000 and 1000 mg/kg bw/day from days 6 to 19 post-coitum.

There were no maternal treatment-related effects in mortality, clinical signs, body weight, food consumption, or cesarean parameters observed in the 100, 300 and 1000 mg/kg/day groups. The maternal NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

There were no treatment-related effects in developmental parameters observed in the 100, 300 and 1000 mg/kg/day groups. The developmental NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

 

There are no data gaps. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.

 

Conclusion

The NOEL of 300 mg a.i./kg bw/day for developmental toxicity and embryotoxicity is derived from the prenatal developmental toxicity study conducted with C8-18 AAPB.

 

There are no data gaps for the endpoint fertility. No human data are available. However, there is no reason to believe that these results from rabbit would not be applicable to humans.

 

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

 

This read-across approach is justified based on structural similarities. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a variety in their carbon chain length, which obviously does not have a relevant impact on acute toxicity as demonstrated by the available data on the source substances.

 

a. Structural similarity and functional groups

The target substance C8-10 Alkylamidopropyl betaine is a UVCB substance manufactured from fatty acids (C8 and C10) and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

The source substance C8-18 AAPB is a UVCB substance manufactured from natural fatty acids or oils with N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance Formamidopropylbetaine is a monoconstituent substance manufactured from formic acid and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

 

b. Differences

Differences in chemical and other intrinsic properties of the target and source substances could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length lead to a rising average lipophilicity as can be seen from the increasing log Kow from Formamidopropylbetaine (log Kow: -3.3), C8-10 Alkylamidopropyl betaine (log Kow: 2.2), C12 AAPB (log Kow: 3.54), C8-18 AAPB (log Kow:4.23).

However, based on the available toxicological data it is demonstrated, that this read-across approach is nevertheless reliable.

 

- Different amounts of unsaturated fatty ester moieties:

The source substance C8-18 and C18 unsatd. AAPB contains considerable amounts of unsaturated C18 chains, which represents a worst case with respect to some toxicological endpoints, mainly local effects (e.g. irritation, sensitisation). But in general, variability in the fatty acid moiety is not expected to be relevant to the intrinsic systemic or developmental toxicity of the substances.

 

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of developmental toxicity data

Endpoints	Source substances		Target substance
	C8-18 AAPB	Formamidopropylbetaine	C8-10 Alkylamidopropyl betaine
Prenatal developmental toxicity	WoE_Developmental toxicity / teratogenicity: 97862-59-4_8.7.2_CESIO_2004_OECD 414 Key study OECD TG 414, rat, oral: gavage NOEL maternal toxicity: 100 mg a.i./kg bw/day NOEL embryotoxicity: 300 mg a.i./kg bw/day NOEL teratogenicity: 1000 mg a.i./kg bw/day Maternal toxic effects: yes Embryotoxic / teratogenic effects: yes   Reliability: 1 (reliable without restriction), GLP	WoE_RA_Developmental toxicity / teratogenicity NOTOX 497620   OECD TG 414, rat, oral: gavage   NOEL maternal toxicity: 1000 mg a.i./kg bw/day NOEL teratogenicity: 1000 mg a.i./kg bw/day   Maternal toxic effects: no Embryotoxic / teratogenic effects: no   Reliability: 1 (reliable without restrictions), GLP	No data, read-across

 

No experimental data are available for the target substance.

In the prenatal developmental toxicity study (tested doses up to and including 1000 mg a.i./kg bw/d) C8-18 AAPB showed no teratogenic activity, and embryotoxic effects were found only at the maternal toxic dose level. The second source substance Formamidopropylbetaine showed no teratogenic or embryotoxic activity.

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The studies were conducted according to OECD Guideline 414 and reliable without restrictions (RL1, GLP).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

Conclusion

Based on structural similarities of the target and source substancesas presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substances C8-18 AAPB and Formamidopropylbetaine are also valid for the target substance C8-10 Alkylamidopropyl betaine.

Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles: low systemic toxicity, not mutagenic, no effects on fertility.

As genotoxicity is one possible mechanism for developmental toxicity, the negative outcome of the genotoxicity tests for the target and source substances further supports the read-across. Genotoxicity is based on covalent binding of the substance itself or reactive metabolites to cellular macromolecules as rate determining step. A similar mechanism based on reactive metabolites is involved in sensitisation; target and source substances were not sensitisers. Thus, the consistency across these endpoints increases the confidence in the conclusion that there is no concern for reactive metabolites.  

Justification for selection of Effect on developmental toxicity: via oral route:
no single study has been selected as key study, instead all available data are considered together in a weight-of-evidence approach

Justification for classification or non-classification

Based on the available data C8-10 Alkylamidopropyl betaine does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to the criteria given in regulation (EC) 1272/2008. Thus, no labelling is required.