1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-18(even numbered) acyl derivs., hydroxides, inner salts

Endpoint summary

Description of key information

Reliable data on repeated dose toxicity of AAPBs are available for the oral route from 28-day and 90-day gavage studies as well as from a 90-day feeding study in rats. In these studies performed according to the corresponding OECD Guidelines on C8-18 AAPB and Coco 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. 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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

Key result

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 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD guideline study, RL1, 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

Reliable data on repeated dose toxicity of C8-18 AAPB are available from a 90-day gavage study in rats as well as from a 28-day gavage study and a 90-day feeding study conducted with the closely related C8-18 and C18 unsatd. AAPB.

A justification for read-across is given below.

 

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, 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/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.

 

Supporting data are available from a subacute study similar to OECD Guideline 407. C8-18 and C18 unsatd. AAPB (Coco AAPB; ca. 30% 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 28 days, 5 d/week.

Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 28 days without treatment to detect recovery from, or persistence of toxic effects.

The reported compound-related findings in the 1000 mg/kg bw group animals (reversible irritative effects at the forestomach) have been interpreted as symptoms of the irritative action of the test article and not as symptoms of a cumulative-systemic toxicity of the test substance.

It was concluded that a daily administration of the test substance up to 1000 mg/kg bodyweight is not cumulative-systemic toxic to 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 rabbit would not be applicable to humans.

 

Justification for read-across

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

This read-across approach is justified based on structural similarities. All AAPBs contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a minor variety in their fatty acid moiety (chain length and degree of unsaturation), which may have an influence on the outcome of skin and eye irritation studies, but is not expected to have any influence on systemic toxicity.

 

a. Structural similarity and functional groups

Alkylamidopropyl betaines (AAPBs) are – with the exception of C12 AAPB - UVCB substances (Substances of Unknown or Variable composition, Complex reaction products or Biological materials), which are defined as reaction products of natural fatty acids or oils with dimethylaminopropylamine and further reaction with sodium monochloroacetate. AAPBs are amphoteric surfactants, which are characterized by both acidic and alkaline properties.

 

Their general structure is:

 

R-C(O)-NH-(CH₂)₃-(N(CH₃)₂)⁺-CH₂-C(O)O^-

R = fatty acid moiety

 

The fatty acids have a mixed, slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 may be included. Consequently, the AAPBs differ by their carbon chain length distribution and the degree of unsaturation in the fatty acid moiety. However, Lauramidopropyl betaine (C12 fatty acid derivate) is the major ingredient of all AAPBs covered by this justification as listed in table 1 “Substance identities” of the general justification for read-across.

 

The substances under evaluation share structural similarities with common functional groups (quaternary amines, amide bonds and carboxymethyl groups), and fatty acid chains with differences in chain length and degree of saturation.

 

b. Differences

Differences in systemic toxicity of the AAPBs 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 could result in a rising average lipophilicity. A variability in the fatty acid moiety is not expected to have any influence on thesystemic toxicityof the AAPBs

- Different amounts of unsaturated fatty ester moieties:

Effects may be expected for e.g. physical state and for some toxicological endpoints, mainly local effects (e.g. irritation). A variability in the fatty acid moiety is not expected to have any influence on the systemic toxicity of the AAPBs.

 

Comparison of repeated dose toxicity data

 

Endpoints	Target substance	Source substance
	C8-18 AAPB	C8-18 and C18 unsatd. AAPB
Repeated dose toxicity, oral	Key_gavage_Repeated dose toxicity: oral: 97862-59-4_ 8.6.2_Goldschmidt_1991_OECD 408 key study OECD TG 408, subchronic, rat, oral: gavage NOEL systemic effects: 300 mg a.i./kg bw/d (highest tested dose, 1000 mg/kg bw/d based on product (a.i. ca. 30 %)) LOEL local effects: 150 mg a.i./kg bw/d (500 mg/kg bw/d based on product (a.i. ca. 30 %)) NOEL local effects: 75 mg a.i./kg bw/d (250 mg/kg bw/d based on product (a.i. ca. 30 %))   Reliability: 1 (reliable without restriction), GLP	Key_feeding_Repeated dose toxicity: 61789-40-0_8.6.2_90days_Unilever_A03_FT890785   Key study   OECD TG 408, subchronic, rat, oral: feed   NOEL effects relevant to humans: 247 mg a.i./kg bw/d (highest tested dose, 1 % in feed, 731 mg/kg bw/d based on product (a.i. 33.8 %))   LOEL: 97 mg a.i./kg bw/day) (0.4% in feed, 288 mg/kg bw/d based on product (a.i. 33.8 %))   Reliability: 1 (reliable without restriction), GLP
	Sup_Repeated dose toxicity: oral: 97862-59-4_8.6.1_Goldschmidt_1991_OECD 408_14-d dose finding   Supporting study 14 d dose range finding study, subacute, rat, oral: gavage NOEL 375 a.i./kg bw/d (highest dose tested, 1250 mg/kg bw/d based on product (a.i. approx. 30 %)   Reliability: 2 (reliable with restrictions), GLP	Sup_Repeated dose toxicity: oral: 61789-40-0_8.6.1_Henkel_1991_OECD 407   Supporting study OECD TG 407, subacute, 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. 30 %)) LOAEL local effects 300 mg a.i./kg bw/d (highest tested dose, 1000 mg/kg bw/d based on product (a.i. 30 %)) NOEL local effects 150 mg a.i./kg bw/d (500 mg/kg bw/d based on product ( a.i. 30%))   Reliability: 1 (reliable without restriction), GLP

 

In these studies performed according to the corresponding OECD Guidelines 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.

Both key studies were conducted according to OECD Guideline 408 and were reliable without restrictions (RL1, GLP). A supporting study conducted according to OECD Guideline 407 (RL1, GLP) is available, as well as a 14 d dose range finding study (RL2, GLP).

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

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

 

Conclusion

Based on structural similarities of the target and source substancesas presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substances C8-18 AAPB as well as C8-18 and C18 unsatd. AAPB are also valid for the target substance C12 AAPB.

The repeated dose toxicity of the whole group of AAPBs is expected to be in the same range as variability in the fatty acid moiety is not expected to be relevant to the intrinsic systemic toxicity of the compounds. Systemic toxicity was low in both 90 d repeated dose toxicity studies:No indication of any systemic toxicity of C8-18 AAPB as well as C8-18 and C18 unsatd. AAPB up to and including the highest tested doses, relevant in view of a potential serious health risk for humans was found.

Justification for classification or non-classification

There is no evidence for intrinsic toxic properties of AAPBs relevant to humans obtained from the results of reliable, relevant and adequate subacute and subchronic oral studies on rats.

Therefore no classification is required for repeated dose toxicity according to CLP, EU GHS (Regulation (EC) No 1272/2008) and directive 67/548/EEC with respect to systemic toxicity.