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Administrative data

Description of key information

- Subacute (14 day dose-range finding study) repeated dose toxicity study oral (gavage), rat Crl:WI(Han)) m/f (similar to OECD TG 407, GLP), dose levels: 0, 50, 200, 500 mg/kg bw/d; no NOAEL derived (dose range finding study): all animals at 500 mg/kg bw/d were sacrificed for humane reasons; slight changes in haematology and clinical biochemistry at 50 and 200 mg/kg bw/d; read-across: Stearic acid 3 -(dimethylaminopropyl)amide

- subacute (28 d study) repeated dose toxicity study oral (gavage), rat Sprague-Dawley m/f (similar to OECD TG 407), dose levels: 0, 30, 100 and 300 mg/kg bw/d (24-26% aqueous solution but it is not clear if the doses mentioned are corrected for undiluted test substance); NOAEL = 75 mg a.i./kg bw/day; read-across: Cetrimoniumchloride

- subchronic (90 d study) repeated dose toxicity study oral (diet), rat Sprague-Dawley m/f (OECD TG 408), dose levels: 0, 100, 500 or 2,000 ppm, corresponding to 0, 22, 113 and 273 mg a.i./kg bw/day; NOAEL = 500 ppm (113 mg a.i./kg bw/d); read-across: C12-18 TMAC (Coco alkyl trimethyl ammonium chloride)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substance have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: quaternary amines, amides, and saturated fatty acid chains with comparable length
• the metabolism pathway leads to comparable products (amine backbone and long chain fatty acids) and non-common products predicted to have no toxicological effects (long chain fatty acids).

Therefore, read-across from the existing genotoxicity studies on the source substance is considered as an appropriate adaptation to the standard information requirements of REACH regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see justification for read-across attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see justification for read-across attached to IUCLID section 13

4. DATA MATRIX
see 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 source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Species:
rat
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
90 d
Key result
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Clinical signs of toxicity, reduced body weight gain, reduced food efficiency and microscopic changes in the spleen and kidneys of high dose animals.
Critical effects observed:
no
Conclusions:
The 90 d NOAEL of the target substance is considered to be 113 mg a.i/kg bw/d.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
113 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available studies were conducted according to guideline and are of high quality.

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

No experimental data on repeated dose toxicity are available for C16 Alkylamidopropyltrimethylammonium Chloride. However, reliable data from an oral subacute repeated dose toxicity study conducted with the closely related source substance Cetrimonium chloride as well as an oral subchronic repeated dose toxicity study conducted with C12 -18 TMAC (Quaternary ammonium compounds, C12-C18 (even numbered) alkyltrimethyl chloride) are available.

Further supporting data are available from a 14 d dose range finding study in rat and an oral reproduction / developmental toxicity screening test in rats, both conducted with the closely related source substance Stearic acid 3-(dimethylaminopropyl)amide. A justification for read-across is given below.

 

A 28-day repeated dose toxicity study was conducted on Cetrimonium chloride in rats according to OECD guideline 407. Groups of 10 male and female rats were administered 0, 30, 100 and 300 mg/kg bw/day test substance by oral gavage for 28 days. There were no treatment-related changes at the 30 and 100 mg/kg bw/day. In the high-dose group there was an increase in water consumption, changes in the absolute and relative weights of the adrenals and spleens (in males) without corresponding effects on haematology, clinical chemistry and histology. The forestomach of the high-dose group showed few microscopic changes, however animals in the high-dose recovery group showed a complete and regular regeneration of the forestomach mucosa. Hence, the forestomach effects were considered to be due to the irritating properties of the test substance rather than symptoms of systemic toxicity. The NOAEL for systemic effects was 300 mg/kg bw/day as test material.

The applied test substance was a 24-26% aqueous solution but it is not clear from the report if the doses mentioned were corrected for active substance. Thus, for safety reasons, this correction was applied resulting in a NOAEL of 75 mg/kg bw/d in terms of active substance.

 

This NOAEL of 75 mg/kg bw/d is also more in line with the NOAEL for general toxicity obtained in the OECD Guideline 421 study conducted with the source substance Stearic acid 3-(dimethylaminopropyl)amide (NOAEL(general toxicity) = 70 mg/kg bw/d based on effects on body weight and food consumption at 200 mg/kg bw/d). This study is discussed in detail in section “Toxicity to reproduction”.

 

Sprague-Dawley rats were administered C12-18 TMAC (0, 100, 500 or 2,000 ppm, corresponding to 22, 113 and 273 mg a.i./kg bw/d) in the diet for 90 days according to OECD guideline 408. The highest dose of 2,000 ppm was reduced to 1,000 ppm from Day 29 onwards due to deterioration in health of the test animals at 2,000 ppm. At the highest dose, the treatment-related findings were clinical signs of toxicity, reduced body weight gain and food efficiency, organ weight changes and microscopic changes in the spleen and kidneys. At the mid dose, reduced body weight gain (males) and reduced food consumption, reduced absolute heart weight and higher incidence of haemosiderin accumulation in the kidneys of males was observed. No treatment-related effects were observed at the lowest dose. Hence, the NOEL was considered to be 100 ppm (i.e., equivalent to 22 mg a.i./kg bw/d). The changes observed at 500 ppm were considered to be minor, isolated effects associated with the reduced palatability of the test substance and were considered not to represent an adverse health effect. The NOAEL was therefore 500 ppm (i.e., equivalent to 113 mg a.i./kg bw/d).

 

In a 14 d dose range finding study according to OECD guideline 407, adopted 03 October 2008, and EU method B.7, May 2008, Stearic acid 3-(dimethylaminopropyl)amide was administered to 3 Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.

All animals in the 500 mg/kg bw/d dose group were sacrificed for humane reasons between days 6 and 8. They showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included: lymphoid atrophy of the thymus, correlating to a reduced size of the thymus at necropsy; hyperplasia and inflammation of the forestomach; hyperplasia of the villi in the duodenum and jejunum; foamy macrophages and sinusoidal dilation and congestion/ erythrophagocytosis in the mesenterial lymph node; absence of spermiation and degeneration of spermatids in the testes, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of week 2 only, whilst no clinical signs were noted at 50 mg/kg bw/d. Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d. The results from this study were used to select the doses for the reproduction/developmental screening study. No reliable NOAEL could be derived from this dose-range-finding study. Due to the small number of animals, the relevance of the clinical biochemistry changes could not be fully justified.

 

Based on the available data, the overall dose descriptor for repeated dose toxicity for the target substance C16 Alkylamidopropyltrimethylammonium Chloride is considered to be 113 mg/kg bw/d.

 

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 and rabbits would not be applicable to humans.

 

Justification for read-across

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

 

Structural similarity

a. Structural similarity and functional groups

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is manufactured from hexadecanoic acid and N,N-dimethyl-propylenediamine. Methyl chloride is used to quaternise the dimethylamino group of the fatty acid amidoamine.

The substance is composed of mainly C16 amides (ca. 92%) of DMAPA and small amounts of the C14 (ca. 2.5%) and C18 amide (ca. 5.5%).

 

The source substance Stearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid and N,N-dimethylpropylenediamine. It is composed of mainly C18 amides (> 89.8%) of DMAPA and small amounts of the C16 amide (<7%).

 

The source substance Cetrimonium chloride is a quaternary ammonium salt manufactured from the tertiary amine N,N-dimethylhexadecanamine and quaternised with Methyl chloride.

 

The target substance C12-18 TMAC is comparable to Cetrimonium chloride, but contains C12-18 chains. More details on chain length distribution are not available for this source substance.

 

 

b. Common breakdown products

The metabolism that is expected to occur is for the target substance C16 Alkylamidopropyltrimethylammonium Chloride and the source substance Stearic acid 3-(dimethylaminopropyl)amide the hydrolysis of the amide-bond by amidases. Metabolism would result in free fatty acids and di- or trimethylaminopropylamine. The free fatty acids enter normal metabolic pathways (e.g. degradation by the mitochondrial beta-oxidation process) and are therefore indistinguishable from fatty acids from other sources including diet.

The amine compounds are not expected to be further metabolised, but excreted via the urine mainly unchanged. 

 

The most likely metabolism of the source substances Cetrimonium chloride and C12-18 TMAC is oxidation of the alkyl chain.

 

c. Differences

C-chain length:

The slight differences in fatty acid chain length (higher percentage of C16 in the target substance vs. higher percentage C18 in the source substance Stearic acid 3-(dimethylaminopropyl)amide) are not considered to be of relevance for systemic toxicty.

The source substance C12-18 TMAC additionally contains shorter C-chains (C12 and C14). However, as fatty acid independent of their chain length are in general not toxic, this difference is not considered to contribute to systemic toxicity. Nevertheless, the bioavailability of C12-18 TMAC may be higher compared to the target substance due to the amounts of shorter C-chains (lower molecular weight and lower lipophilicity are in general more favourable for uptake). Therefore, C12-18 TMAC may be considered to represent a worst case.

 

Methylation/quaternation:

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is methylated during the manufacturing process resulting in the quaternised ammonium ion.

Stearic acid 3-(dimethylaminopropyl)amide)on the other hand is not methylated during the manufacturing process. But based on physicochemical data (pKa) it is concluded that at physiological relevant pH, the substance is mostly protonated similarly resulting in a positively charged ammonium ion. This difference isnot considered to be of relevance forsystemic toxicity.

 

Amide:

In contrast to the source substances Cetrimonium chloride and C12-18 TMAC, the target substance C16 Alkylamidopropyltrimethylammonium Chloride as well as the source substance Stearic acid 3-(dimethylaminopropyl)amide) contain a polar amide function which may be susceptible to enzymatic hydrolysis. However, the available repeated dose toxicity studies conducted with the source substances Cetrimonium chloride and C12-18 TMAC on the one hand and the source substance Stearic acid 3-(dimethylaminopropyl)amide) on the other hand demonstrate, that this structural difference has no important effect on the outcome of the studies.

 

Comparison of repeated dose toxicity data

Endpoint

Target substance

C16 Alkylamidopropyltrimethylammonium Chloride 

Source substance

Stearic acid 3-(dimethylaminopropyl)amide

Cetrimonium chloride

C12-18 TMAC

Repeated dose Toxicity

 

No data, read-across

No data

NOAEL = 300 mg/kg bw/day as test material, corresponding to 75 mg a.i./kg bw/d(24-26% aqueous solution)

 

reversible forestomach effects in the highest dose group were considered to be due to the irritating properties of the test substance

 

Similar to OECD TG 407, rat, oral (gavage), RL 1, GLP

 

Dose levels:0, 30, 100 and 300 mg/kg bw/day asa 24-26% aqueous solution (not clear if dose levels were corrected for active substance)

 

post-exposure recovery period in satellite groups: at least 27 d

NOAEL = 113 mg a.i./kg bw/day

 

clinical signs of toxicity, reduced body weight gain and food efficiency, organ weight changes and microscopic changes in the spleen and kidneys at 273 mg a.i./kg bw/d

 

OECD TG 408, rat, oral (diet), RL 1, GLP

 

Dose levels: 0, 100, 500 and 2000 ppm (reduced to 1000 ppm from day 29), equivalent to 0, 22, 113 and 273 mg a.i./kg bw/d

 

 

Supporting data

No data, read-across

OECD guideline 421(Reproduction/Developmental Toxicity Screening Test)
RL 1, GLP)

10 Wistar rats/sex/dose exposedby gavageat dose levels of 0, 20, 70 and 200 mg/kg bw/d.

Males were exposed for 28 days; Females were exposed for 41 – 54 days

 

General toxicity: reduced body weight gain and food intake at 200 mg/kg bw/d

 

parental NOAEL: 70 mg/kg bw/d

 

 

14 d dose range finding study similar to OECD TG 407,rat, oral (gavage), RL 1, GLP

 

500 mg/kg bw/d: all animals of the highest dose group were sacrificed for humane reasons based on clinical signs (forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach)

 

In the 50 and 200 mg/kg bw/d dose groups, no mortality occurred, and no or only minor clinical signs were observed. No treatment related effects were observed at necropsy or histopathology.

 

50 and 200 mg/kg bw/d: Clinical biochemistry changes (higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d)

 

 

The lower molecular weight of the source substances Cetrimonium chloride and C12-18 TMAC, the lower log Kow and the expected higher water solubility of C12-18 TMAC may lead to a higher bioavailability and therefore, both source substances can be regarded as worst case for repeated dose toxicity.

Based on the structural similarities with identical functional groups (quaternary amines) and saturated fatty acid chains with comparable length as well as similar physicochemical properties, the read-across approach from the available repeated dose toxicity studies is considered to be reliable.

 

Quality of the experimental data of the analogues:

The source substance Cetrimonium chloride has been tested in a reliable study comparable to OECD TG 407.

The source substance C12-18 TMAC has been tested in a reliable study according to OECD TG 408.

The source substance Stearic acid 3-(dimethylaminopropyl)amide has been tested in a reliable study comparable to OECD TG 407 (dose range finding study) as well as in a reliable study according to OECD TG 421.

All tests have been conducted according to GLP criteria. Therefore these data have no uncertainties and can be used in an analogue approach. The available data from the source chemical is sufficiently reliable to justify the read-across approach.

 

Conclusion for read-across

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles under the experimental conditions in the considered studies for the endpoint repeated dose toxicity.

Thus, the dose descriptor for repeated dose toxicity obtained with the source substance C12-18 TMAC is considered to be also relevant for the target substance C16 Alkylamidopropyltrimethylammonium Chloride.

The remaining uncertainty associated with this read-across approach is addressed by the application of an additional assessment factor.

 

 

 

Justification for classification or non-classification

Based on the available relevant and reliable data from the source substances Stearic acid 3-(dimethylaminopropyl)amide, Cetrimonium chloride and Coco alkyl trimethyl ammonium chloride, the target substance C16 Alkylamidopropyltrimethylammonium Chloride does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to repeated dose toxicity.