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

Endpoint summary

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key 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:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 3 October 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 oral 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

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

Remarks:

Doses / Concentrations:
0, 100, 300, 500 mg/kg bw/d
Basis:
actual ingested

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.

Observations and examinations performed and frequency:

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

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: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, prior to necropsy / at the end of Week 2 of the recovery period
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, prior to necropsy / at the end of Week 2 of the recovery period
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 of treatment and once during Week 2 of recovery
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

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

Other examinations:

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.
Moreover, 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.

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.

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.

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

Description (incidence and severity):

Dosing phase
Lymphocytosis and/or monocytosis were recorded in two females dosed at 300 mg/kg bw/day and 3 females receiving 500 mg/kg bw/day. When compared with mean control data, individual changes were between 61% and 93% for lymphocytes and 137% and 150% for monocytes.
The statistically significant increase of eosinophils observed in females dosed at 300 mg/kg bw/day was not dose-related, therefore considered incidental.

Recovery phase
After the recovery period, one female showed lymphocytosis and neutrophilia. When compared with mean control data, the individual change was of 49% and 3.7 fold, respectively. Concerning the other treated animals, lymphocytes showed complete reversibility and monocytes data were comparable with controls, even though values were similar to those observed during the dosing phase.
The statistically significant differences recorded between control and treated animals (reticulocytes and lymphocytes relative count in males, mean corpuscular haemoglobin in females) were not observed during the dosing phase, therefore considered unrelated to treatment.
No changes in coagulation parameter were seen in control and treated animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Chloride and sodium showed a statistically significant decrease in females dosed at 300 and/or 500 mg/kg bw/day, when compared to controls. Due to the minimal severity (up to 2%), these findings were considered of no toxicological relevance.
The statistically significant changes recorded in animals dosed at 100 and/or 300 mg/kg bw/day (bilirubin, bile acids and inorganic phosphorus in males; urea in females) were not dose-related, therefore considered unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant differenecs between treated and control groups were seen in landing foot splay.
Changes in grip strength were considered incidental and not treatment-related, since the direction of changes was opposite in the two sexes: decreases in males and increases were seen in females, receiving 500 mg/kg bw/day, when compared to controls.
No differences between treated and control groups were evident at the motor activity measurements.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant differences were seen in terminal body weights of treated animals respect to controls.
No toxicological relevance was attributed to the statistically significant increase in absolute (13%) and/or relative (9-10%) kidneys weight seen in females treated at 300 and/or 500 mg/kg/day not to the statistically significant increase in relative (up to 7%) kidneys weight seen in males treated at 300 and 500 mg/kg/day, when compared to controls, since these increases were slight and no changes were detected at the histopathological evaluation.
No other differences were seen in treated groups when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant changes were noted in treated animals. The sporadic changes observed in few treated animals could be considered incidental.

Neuropathological findings:

not examined

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. 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

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

Conclusions:

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

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).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990-07-24 to 1990-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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 oral 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, 20g/800 ml, 40g/800 ml, 80g/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: 0g/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

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

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

Observations and examinations performed and frequency:

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals of group 1 (0 mg/kg/bw) and 4 (1000 mg/kg/bw) were examined once predose and during final week of treatment. Group 2 (250 mg/kg/bw) and 3 (500 mg/kg/bw) 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

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

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 per cent 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.

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.

Clinical signs:

no effects observed

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:

no mortality observed

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.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- There were no treatment-related ocular findings.

Haematological findings:

no effects observed

Description (incidence and severity):

-There were no treatment-related hematological changes in animals of either sex.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Although statistical analysis revealed a few significantly different values between the tested groups, there were no treatment-related changes in clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

- Although there was an increased incidence of positive reactions for hemoglobin concentration in the urine of high dose males exclusively, these findings are considered to be of minor biological significance since these results could not be confirmed by microscopic urine analysis.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- Examination of absolute and relative organ weights including weights of ovaries and testes did not reveal any treatment-related changes in male and female animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Macroscopic necropsy findings revealed some stomach ulcer at fundus and cardia region in one high dose male and female each.
There were no other treatment-related macroscopic necropsy findings.

Neuropathological findings:

not examined

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.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

systemic effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no systemic effects

Dose descriptor:

LOEL

Remarks:

local effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: local irritative effects at the side of application (forestomach gastritis), judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans

Dose descriptor:

NOEL

Remarks:

local effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: local irritative effects at the side of application (forestomach gastritis), judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans

Critical effects observed:

no

Conclusions:

Up to and including the highest tested dose of 300 mg a.i./kg bw/d ( = 1000 mg product (a.i. ca. 30 %)/kg bw/d, C8-18 AAPB was tolerated with no evidence of any systemic toxicity in this 90 day rat gavage study.
The only intolerance seen at the mid and high dose level was a local irritative effects at the side of application (forestomach gastritis). This substance related finding is judged as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans. Therefore the NOEL relevant to human DNEL calculation is the 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.

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). The severity of the forestomach gastritis was judged by the pathologist as minimal to moderate. Forestomach gastritis is a common finding in rat gavage studies on irritative test items. This treatment related finding is generally forced by the gavage exposure regime with constantly repeated bolus ingestion and missing when the test items are applicated via food or the drinking water. The reversibility of AAPB induced rat forestomach gastritis and its missing when the test item is applicated via food have been proven in a subacute gavage study with recovery group (see study 61789-40-0_8.6.1_Henkel_1991_OECD 407) and in a subchronic food study (see study 61789-40-0_8.6.2_90days_Unilever_A03_FT890785, both studies also reported in this IUCLID), respectively. A forestomach or a functional correlate to the rat forestomach is missing in humans.The irritative rat forestomach gastritis is judged as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans. 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 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 October 2011 - 19 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: - Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No. 2) and ME (No. 031121002).

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 oral 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

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 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with PC-2011-350 by daily gavage in the rat (NOTOX project 497623).

Positive control:

No.

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

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 when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the 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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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 toxicity was observed up to the highest dose level tested.

Critical effects observed:

not specified

Conclusions:

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

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

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

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-11-17 to 1990-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 oral 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

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

Dose / conc.:

0.04 other: %, nominal in diet

Dose / conc.:

0.1 other: %, nominal in diet

Dose / conc.:

0.4 other: %, nominal in diet

Dose / conc.:

1 other: %, nominal in diet

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

Observations and examinations performed and frequency:

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.

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

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

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 100g 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.

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.

Clinical signs:

no effects observed

Description (incidence and severity):

- All rats showed 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

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- Statistically significant lower food conversion efficiencies were observed over the 13 weeks of treatment for both male and female rats fed 1.00% test item and the female rats fed 0.10% test item. These statistically significant differences were not observed in the recovery rats in the 5 week recovery period.

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

no effects observed

Description (incidence and severity):

- No statistically significant differences in water intakes were observed over the 13 weeks of treatment for either male or female rats fed the test item when compared with controls

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- No treatment related ophthalmological effects were noted.

Haematological findings:

no effects observed

Description (incidence and severity):

- Some statistical significances were observed but these appeared randomly spread with no evidence of a dose-related response. It was considered that there were no direct effects that could be related to the feeding of the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Main study groups:
- Rats fed the test item showed statistically significant differences from the control rats for the following plasma constituents. These observations largely reflect the reduced food conversion efticiency values recorded for these rats.
--I: Reduced plasma 5-nucleotidase for female rats fed 1.00% .
-- II:Reduced plasma triglyceride for male rats fed 1.00%, which was considered a marginal effect.
-- III: Reduced plasma urea for female rats fed 1.00%, which was considered a marginal effect.
Recovery groups:
These statistically significant differences were not observed in the recovery rats at Week 18.

Urinalysis findings:

no effects observed

Description (incidence and severity):

- Some statistically significant differences were observed but these appeared randomly spread with no evidence of a dose-related response. It was considered that there were no direct effects that could be related to the feeding of the test item.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main study groups:
- The following statistically significant differences (P = 0.05) were observed, there was no histological correlate for these organ weight changes.
-- I: Reduced necropsy body weights for female rats fed 0.10% test item. This was considered a marginal effect. The reason for this reduced values is unclear as there was no evidence of a dose-related response.
-- II: Reduced heart and relative heart weights for female rats fed 0.10% test item. The reason for this reduced values is unclear as there was no evidence of a dose-related response.
-- III: Increased caecal and relative caecal weights for both male and female rats fed 1.00% test item.
-- IV: Increased empty caecal weights for male rats fed 1.00% test item, a pooled analysis of male and female rats fed 1.00% test item and
male rats fed 0.40% test item. The latter was considered to be a marginal effect.
-- V: Increased relative empty caecal weights for both male and female rats fed 1.00% test item, and for a pooled analysis of these male and female rats. Increased relative empty caecal weights were also observed for the female rats fed 0.10% test item (marginal effect), which was considered to be related to the reduced post mortem body weights observed for these rats (See I above).
-- VI: Reduced liver weights for female rats and for a pooled analysis of male and female rats fed 1.00% test item. Reduced liver weights were also observed for the female rats fed 0.10% test item (See (I) and (II) above)
-- VII: Reduced relative liver weights for both male and female rats fed 1.00% test item, and for a pooled analysis of these male and female rats.
The significant finding for the male rats alone was considered to be a marginal effect.
-- VIII: Increased testes weights for male rats fed 0.40% test item. This was considered to be a marginal effect.
Recovery groups:
- These statistically significant differences were not observed in the recovery rats at Week 18.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main study groups:
- The abdominal fat depots were reduced in both male and female rats fed 1.00% test item when compared with the control animals. This feature corresponded to the decreased food efficiency values recorded for these rats.
- Enlargement of the caecum was noted at necropsy in 8/12 male rats and 7/12 female rats fed 1.00% test item, with some showing alteration of the colour of the contents from grey-green to green.
Recovery groups:
- An enlargement of the caecum was not observed in the recovery rats at Week 18 apart from a slight enlargement in one female recovery rat. It is considered that slight enlargement of the full caecum can occur as part of the background pathology of the Colworth Wistar rat and may account for this finding in one female rat fed 0.04% test item at Week 13 and one female recovery rat.

Neuropathological findings:

not examined

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.

Histopathological findings: neoplastic:

not examined

Details on results:

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- Analyses of diet obtained from various sites during mixing and preparation indicate that the test item at dietary levels of 0.10%. 0.40% or 1.00% was distributed uniformly in the diets and was stable in diet when stored for up to 2 weeks in either the cold room or the animal room. It is considered
that the 0.04% dietary level is covered by the fact that it was prepared by a 1:10 dilution of the 0.40% dietary level with
control MODAIN purified diet.

PROTEIN AND PROTEINE ELECTROPHORESIS
Main study groups:
- Statistically significant lower serum total protein levels were observed for both male and female rats fed 1.00%. These observations largely reflect the reduced food conversion efficiency values recorded for these rats.
Recovery groups:
These statistically significant differences were not observed in the recovery rats at Week 18.

Key result

Dose descriptor:

NOEL

Remarks:

effects relevant to humans

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see "remark"

Dose descriptor:

LOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on transient effects on food conversion efficiencies and organ weights of caecum and liver, judged as not relevant to humans in view of an potential serious health risk due to missing histopathological correlate and proved reversibility.

Critical effects observed:

no

Conclusions:

Up to and including the highest tested dose of of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day), Coco AAPB was tolerated with no evidence of any systemic toxicity relevant in view of an potential serious health risk for humans in this 90 day rat feeding study with 38 day recovery.

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).

The LOEL is 0.4% in feed (corresponding to 288 mg product/ kg bw/d and 97 mg a.i./kg bw/day),based on transienteffects on food conversionefficiencies and organ weights of caecum and liver, judged as not relevant to humansin view of a potentially serious health riskdue to missing histopathological correlate and proved reversibility.

Endpoint conclusion

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).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the assessment of repeated dose toxicity, a subacute 28 d repeated dose toxicity study is available for the target substance C8-10 Alkylamidopropyl betaine. Further reliable data on repeated dose toxicity are available from 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.

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 a subchronic toxicity study according OECD guideline 408 (1991), 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). The severity of the forestomach gastritis was judged by the pathologist as minimal to moderate. Forestomach gastritis is a common finding in rat gavage studies on irritative test items. This treatment related finding is generally forced by the gavage exposure regime with constantly repeated bolus ingestion, normally reversible after cessation of treatment and almost missing when the test items are applicated via food or the drinking water. The reversibility of AAPB induced rat forestomach gastritis and its missing when the test item is applicated via food have been proven in a subacute gavage study with recovery group (Cognis, 1991) and in a subchronic feeding study (Unilever, 1994), respectively. A forestomach or a functional correlate to the rat forestomach is missing in humans. The irritative rat forestomach gastritis is judged as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans.

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/day (= 1000 mg product (a.i. ca. 30%)/kg bw/day.

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.

 

In a further subchronic toxicity study according OECD guideline 408, C8-18 and C18 unsatd. 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 a 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/day 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 % test item 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 % test item. These animals fed 1.00 % test item 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 alterations, the rat caecum changes 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/day and 247 mg a.i./kg bw/day).

The LOEL is 0.4 % in feed (corresponding to 288 mg product/ kg bw/day and 97 mg a.i. /kg bw/day), based on transient effects on food conversion efficiencies and organ weights of caecum and liver, judged as not relevant to humans in view of a potentially serious health risk due to missing histopathological correlate and proved reversibility.

 

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.

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

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

 

Conclusion

The NOEL for systemic effects relevant to human DNEL calculation is derived from the 90 d repeated dose toxicity study with C8-18 AAPB which is the highest tested dose of 300 mg a.i./kg bw/day (= 1000 mg product (a.i. ca. 30%)/kg bw/day.

 

There are no data gaps for the endpoint repeated dose toxicity. No human data are available. However, there is no reason to believe that these results from rat 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 repeated dose toxicity as demonstrated by the available data on the target and source substances.

 

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, including unsaturated C18 chains.

 

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

 

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).

There are clear trends in the physicochemical properties (with regard molecular weight, water solubility, log Kow and surface tension) as demonstrated in detail in the general justification for read-across. As data on repeated dose toxicity are available for the taget substance (subacute study) as well as for the upper and lower end of this row similarly showing low systemic toxicity, the differences in C chain lengths obviously do not influence the intrinsic toxicity. 

- 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).

 

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

 

Comparison of repeated dose toxicity data

 

Target substance	Source substances
C8-10 Alkylamidopropyl betaine	C8-18 and C18 unsatd. AAPB	C8-18 AAPB	Formamidopropylbetaine
WoE_Repeated dose toxicity: 73772-45-9 / 73772-46-0_8.6.1_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	WoE_RA_feeding_Repeated dose toxicity: 61789-40-0_8.6.2_90days_Unilever_A03_FT890785   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 restrictions), GLP	Key_RA_gavage_Repeated dose toxicity: oral: 97862-59-4_ 8.6.2_Goldschmidt_1991_OECD 408 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 restrictions), GLP	WoE_RA_Repeated dose toxicity: 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

 

The 28 day NOAEL for C8-10 Alkylamidopropyl betaine was 500 mg a.i./kg bw/d (highest tested dose level).

In the subchronic toxicity study (according to OECD Guideline 408) with Formamidopropylbetaine 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 were reported. A NOAEL of at least 1000 mg a.i./kg was established.

In the studies on C8-18 AAPB and C8-18 and C18 unsatd. AAPB, up to and including the highest tested doses, no indication of any systemic toxicity of AAPBs relevant in view of a potential serious health risk for humans was found.

The only treatment related effect seen in the gavage studies was a local inflammatory response at the site of application (forestomach gastritis) most probably caused by an irritant effect of the test item.

Reversibility of the forestomach gastritis was shown in the 28-day gavage study.

In the 90 d feeding study transient effects on food conversion efficiencies and organ weights of caecum and liver were observed, but judged as not relevant to humans in view of a potentially serious health risk due to missing histopathological correlate and reversibility.

 

The NOELs derived from the 90-day gavage and the 90-day feeding study relevant in view of a potential serious health risk for humans were the highest tested doses of 300 mg a.i./kg bw/day (corresponding to 1000 mg product (a.i. ca. 30%)/kg bw/day) and 1% in feed (corresponding to 731 mg product/kg bw/day and 247 mg a.i./kg bw/day based on measured food consumption), respectively.

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 407 or 408 and were 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, 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.

The source substances were of low systemic toxicity in the available subchronic repeated dose toxicity studies. Based on the absence of toxicologically relevant effects up to the highest dose level tested of 500 mg/kg bw/d in the subacute toxicity study, similarly low systemic toxicity after subchronic exposure is also expected for the target substance C8-10 Alkylamidopropyl betaine. 

Justification for classification or non-classification

Based on the available data, C8-10 Alkylamidopropyl betaine  does not need to be classified for specific target organ toxicity after repeated exposure according to regulation (EC) 1272/2008. Thus, no labelling is required.