1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route was requested with the analogue substance (carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide (C12 AAPB, CAS no 4292-10-8, EC no 224-292-6).

Since no systemic toxicity studies were available for the analogue substance so far, preliminary studies were initiated, including a Reproduction/Developmental toxicity screening test in rats (OECD TG 421), which was supposed to provide the necessary information to select appropriate dose levels for the subsequent Extended One-Generation Reproductive Toxicity Study (OECD TG 443).

However, due to unexpected mortality and adverse effects at the very beginning of the treatment period (mortality of 8/10 males and 3/10 females in the high dose group and severe local effects in the stomach in mid dose group in 6/10 males, 10/10 females), it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.
Therefore, in agreement with the Sponsor, it was decided to interrupt the treatment at 1000 mg/kg/day from Day 3 of the study and to investigate only the toxicity of the test item, C12 AAPB, changing the study design of the OECD TG 421 study to a 2-week oral preliminary toxicity study.
Additional studies were initiated to further investigate the reasons for this unexpectedly high local toxicity and to determine suitable dose levels for the OECD TG 421 and subsequent OECD TG 443 study.

Further details on the delay are attached below.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD guideline studies, RL1, GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A preliminary toxicity study was conducted on the source substance C12 AAPB as requested by ECHA: Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley rats of both sexes after repeated dosing with C12 AAPB, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring in agreement with the OECD guideline 421. The animals, 10 per sex, were assigned to four groups and administered orally, by gavage, at dose levels of 100, 300 and 1000mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10mL/kg.

Due to unexpected mortality and adverse effects at the very beginning of the treatment period, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.

Therefore, in agreement with the Sponsor, it was decided to interrupt the treatment at 1000 mg/kg/day from Day 3 of the study and to investigate only the toxicity of the test item, C12 AAPB, changing the current study design to a 2-week oral preliminary non GLP study.

 

Mortality

Eleven cases of premature death, 8 males and 3 females receiving 1000mg/kg/day (Group 4) were found dead on Day 2 of treatment or, in one instance (one male), sacrificed for humane reasons on Day 3 of treatment.

Ataxia, decreased activity, hunched posture (kyphosis), staining of the ventral region, piloerection and salivation were the treatment-related clinical signs recorded in all these animals just prior their deaths. At the post mortem examination, yellow, brown and/or red staining of ventral or urogenital region and/or muzzle and dark and/or red colour of brain were observed. All the reported macroscopic observations were considered contributory to the cause of deaths.

 

Clinical signs

Piloerection and salivation were the two most relevant treatment-related clinical signs recorded in male and female animals dosed at 300mg/kg/day. The signs were considered treatment related but not adverse.

 

Body weight and body weight gain

Changes noted in body weight gain in groups 2 and 3 were considered incidental or not adverse.

 

Food consumption

No differences in food consumption were recorded during the study in animals of Groups 2 and 3, when compared to controls.

 

Terminal body weight and organ weights

No changes were observed in terminal body weight, or absolute and relative organ weights of control and treated animals that completed the treatment or in animals of Group 4 after 14 days of the recovery period.

 

Macroscopic observations

The most relevant change observed in rats treated at 300 and 1000mg/kg/day of both sexes was thickened stomach (non glandular region), when compared to controls.

 

Based on the results obtained in this study, it can be concluded:

– The dose level of 1000 mg/kg/day induced an acute toxicity when administered to Sprague Dawley rats as demonstrated by mortality and severe signs such ataxia, hunched posture and decreased activity. Treatment related findings described as thickened stomach (non glandular region), were observed in animals after 12 days of recovery period.

– The dose level of 300 mg/kg/day induced treatment related clinical signs such as piloerection and salivation, reduction in body weight gain and thickened stomach (non glandular region) observed at the macroscopic examination. These signs were considered treatment-related, but not adverse.

 

The dose level of 300 mg/kg/day is considered the NOAEL (No Observed Adverse Effect Level) for this study.

 

 

Conclusion

When the study data are available, a robust study summary will be prepared and submitted within an update of the dossier. Evaluation will be reconsidered based on the outcome of the extended one generation reproduction toxicity study.

 

 

Effects on developmental toxicity

Description of key information

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

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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: supporting information

Species:

rabbit

Remarks on result:

not measured/tested

Abnormalities:

not examined

Remarks on result:

not measured/tested

Abnormalities:

not examined

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

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: supporting information

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOEL

Remarks:

embryotoxicity

Effect level:

300 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: based on reduced mean fetal weight and increased number of resorptions at 1000 mg a.i./kg bw/day

Dose descriptor:

NOEL

Remarks:

teratogenicity

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no test item-related increase in the incidence of malformations and variations at any of the tested doses, not even at materno-toxic doses

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active ingredient/kg bw/day).
The NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active ingredient/kg bw/day).
The NOEL for external, skeletal or soft tissue malformations and variations was the highest tested dose of 3300 mg/kb bw/day (corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD guideline study, RL1, GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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

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

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

The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active ingredient/kg bw/day).

The NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active ingredient/kg bw/day).

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

 

In HERA risk assessment report first edition, 2005 an additional developmental toxicity study is reported as follows: “One further developmental toxicity study is available with cocamidopropyl betaine (30% active substance). Female pregnant rats were administered 0, 30, 90 or 300 mg/kg bw on days 6 through 17 of gestation. No treatment-related effects on the incidence of fetal external, visceral, or skeletal malformations or developmental variations were observed among litters from dams in any of the treated groups.

The maternal and developmental no-observed-effect level (NOEL) of this study was 300 mg/kg bw/d, the highest level (Colgate-Palmolive, 2000).”

 

 

A 2 week preliminary toxicity study with C12 AAPB was conducted in New Zealand White (NZW) female rabbits with particular aim to investigate its potential local gastric irritating effect.

Female rabbits (4/dose) received the test item, dissolved in softened water, at dose levels of 100, 300 and 600 mg/kg/day (in terms of test item corrected for purity (30.43%)) by oral gavage. The dose volume was set at 5mL/kg. Control animal received softened water as vehicle during the same treatment period. The animals were checked twice a day for mortality and daily monitored for clinical signs. The bodyweight and food consumption were measured twice a week. Detailed macroscopic examination, organ weights and histopathology of the gastrointestinal tract, were carried out.

 

Mortality

On Day 3, two females at 600mg/kg/day, were found dead and the remaining females were sacrificed for humane reasons. On Day 5, one female at 300mg/kg/day was found dead and the remaining females were sacrificed for humane reasons.

At necropsy, the findings affecting the stomach could be considered the factor contributory to the ill status or death of these animals.

No mortality occurred at 100mg/kg/day.

 

Clinical signs

Severe clinical signs including dyspnoea, pale appearance, red nasal discharge, coldness to touch, brown staining in the perianal region and decreased activity were recorded at 300 and 600mg/kg/day. Reduced or liquid faeces were observed in the cage.

Reduced faeces and reduced food intake were recorded at 100mg/kg/day. Dyspnoea, transparent nasal discharge, rales and emaciated appearance were also observed in one low dose female.

 

Body weight

On Day 5, marked reduced body weight was observed in females dosed at 300mg/kg/day (-15% respect to Day 1). Body weight was reduced at 100mg/kg/day, especially in two females.

 

Food consumption

Severe reduced food consumption was noted at 300mg/kg on Day 5. A decrease in food consumption was recorded at 100mg/kg/day starting from Day 5 onward.

 

Terminal body weight and organ weights

A slight decrease was observed in terminal body weight of low dose females sacrificed at term when compared to controls. The statistically significant decrease in absolute and relative uterus weight was considered of doubtful interpretation, as not corroborated by histopathological evaluation.

 

Macroscopic observations

Treatment-related changes were noted in the pyloric region of the stomach of a single low dose female rabbit and was characterised by the presence of multiple pinpoint red areas associated with a single depressed dark red area. The macroscopic changes correlated at histopathological examination with erosion, haemorrhage and chronic inflammation.

 

Microscopic observations

At final sacrifice, treatment-related findings were seen in the stomach of one low dose female and consisted of erosion of the mucosal epithelium associated with haemorrhage and chronic inflammation of tunica muscularis and/or submucosal of fundic region of the stomach.

 

Conclusion

In conclusion, based on the results obtained in this investigative study, signs of toxicity were seen at all dose levels. The treatment-related findings at histopathological examination of the stomach confirmed the potential gastric irritant effect of C12 AAPB.

 

Based on the results of this study, the potential maximum dose level of C12 AAPB for a prenatal developmental toxicity study in rabbit would be 100 mg/kg bw/d or even lower. This is no meaningful highest dose level for an otherwise relatively non-toxic substance.

 

In addition, Braakhuis et al. (2019) found, that potential interspecies differences in developmental N(L)OAEL are in the same range as the interstudy reproducibility error, making the added value of a study conducted in a second species questionable. The authors have analysed a database with rat and rabbit developmental toxicity studies for over 1000 industrial chemicals, pesticides, veterinary drugs and human pharmaceuticals, which included 143 compounds with multiple oral rat studies and 124 compounds with multiple oral rabbit studies. This analysis demonstrated, that on average over all compounds, rat and rabbit do not differ in sensitivity towards developmental effects.

 

Reference:

Braakhuis HM et al. (2019) Testing developmental toxicity in a second species: are the differences due to species or replication error? Regulatory Toxicology and Pharmacology 107 (2019) 104410

 

There are no data gaps for the endpoint developmental 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 classification or non-classification

There is no evidence for an intrinsic toxicity to reproduction of AAPBs from the results of an reliable oral developmental toxicity / teratogenicity study on rats at doses up to and including the guideline limit dose of 1000 mg a. i./kg bw/day and reliable oral subchronic and subacute repeated dose toxicity studies with histopathological examination of the male and female reproductive organs (epididymides, testes, seminal vesicle, prostate, ovaries, fallopian tubes, uterus, vagina, mammary gland).

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