Amines, C12-18-alkyldimethyl, N-oxides

Endpoint summary

Description of key information

The key study for the oral route is a 90-day repeated dose oral toxicity study in rats comparable to OECD TG 408 [Hazelton Laboratories (1974)] performed with C12-14 AO. The NOAEL was 0.1% (in the diet) or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain and age, this translates into a delivered dose of 88 mg AO/kg bw/day. This value represents the highest NOAEL below the lowest LOAEL and was selected for use in the risk assessment to characterize the risk of long term systemic toxicity via the oral and (by route to route extrapolation) dermal and inhalation routes.
With regard to dermal toxicity, repeated dermal treatment of rats (6 hours/day/5 days/week) for 90 days with C12-14 AO at dosage levels of 0.27 % AO and 1.33 % AO revealed local signs of irritation but no effects attributable to direct systemic toxicity. A NOAEL regarding systemic effects was therefore not established. Under the conditions of the study the LOEL for local dermal toxicity (irritation) in mice was determined to be 0.27 % AO. Based on application of 0.27 mg AO via a patch of size 2x3 cm, this is equivalent to 0.045 mg AO/cm².

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

88 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

There are six reliable (Klimisch score = 2) studies performed using C12-14 AO. In addition, there are reliable (Klimisch score =1) OECD 422 studies for both C12-14 AO and C12-18 AO.

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 adverse effect observed

Quality of whole database:

Two reliable studies available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

0.045 mg/cm²

Study duration:

subchronic

Species:

mouse

Additional information

Repeated dose toxicity oral:

Repeated dose toxicity of C12 -18 AO has been examined in an OECD TG 422 study [Hansen B (2010)]. In this study rats (CD/Crl:CD(SD); 10 animals/sex/group) were dosed by oral gavage with C12 -18 AO at levels of 0, 40, 100 or 200 mg AO/kg bw/day for two weeks prior to mating, during mating and approximately two weeks after mating (males) and for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice for females. A satellite group of non-mated animals was treated in the same manner as the main study animals and kept without treatment at least 14 days after the first scheduled sacrifice of main study females. No test item related mortality was noted in any group. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg AO/kg bw/day. Increased salivation was noted for a few male and females at 100 mg AO/kg bw/day and for most animals at 200 mg AO/kg bw/day during all periods of the study starting at test day 1. Further, at 200 mg AO/kg bw/day laboured breathing was observed for one male and rough fur was noted in several females during the pre-mating, mating, gestation and/or lactation period. In main study animals, no influence was noted on the parameters of the functional observations at any of the tested dose levels for males. Females treated with 200 mg AO/kg bw/day revealed salivation and piloerection. In addition, hind limb grip strength of the females was reduced (p<=0.01) by up to 71 % starting at the 40 mg AO/kg bw/day group though no dose response relationship was noted. A slight but not significant reduction was also observed for the males at 100 and 200 mg AO/kg bw/day. No influence was noted on the bodyweight of main study and satellite animals during the entire study at any dose. No test item related influence was observed on food consumption of the males and female animals treated with either 40 or 100 mg AO/kg bw/day. Treatment with 200 mg AO/kg bw/day resulted in a food intake statistically significantly reduced by 11 % in test week 2 for main study males and for the female main study animals by 10 % in week 1 and by 13 % on gestation day 7 compared to the control. The food intake of the female satellite animals treated with 200 mg AO/kg bw/day was statistically significantly reduced by 20 % in week 1 compared to controls. No effect on water consumption was noted at any of the tested dose levels. No test item related changes in haematological parameters were noted at the end of the premating period (day 15) of male and female rats treated with 40 or 100 mg AO/kg bw/day compared to controls. In 200mg AO/kg bw females there was a statistically significant (p <= 0.01) in Neut (absolute) compared to controls. No test item related changes were noted on biochemical parameters on test day 15 of female rats treated with 40 mg AO/kg bw/day compared to the control. ALAT levels were significantly increased (p<= 0.01) in 200 mg AO/kg bw/day males and females. Macroscopic inspection at necropsy revealed no test item related changes in organs or tissues at any dose. No effect on organ weights was noted. Microscopic examination was restricted to 5 animals/sex of the control and 200 mg AO/kg bw/day groups, all satellite animals and the forestomach and mesenteric lymph node of 5 animals/sex of the 40 and 100 mg AO/kg bw/day groups. The forestomach revealed a squamous cell hyperplasia with submucosal inflammatory reaction and hyperkeratosis/parakeratosis in the stratum corneum of the animals treated with 200 mg AO/kg bw/day. These lesions were completely reversible within the 16 days recovery period. Further, a dose dependent increase of macrophages with vacuolisation in the mesenteric lymph nodes was observed in the animals treated with 100 or 200 mg AO/kg bw/day. The effect was still noted at the end of the recovery period. The NOEL from this study was 40 mg AO/kg bw/day.

In a similar study performed using C12 -14 AO [Ceccatelli R (2008)] rats (HanRcc: WIST(SPF)) were dosed by oral gavage with C12 -14 AO (Ammonyx LO)) at levels of 40, 100 and 250 mg AO/kg bw/day for at 28 days (males) and for 14 days prior to pairing, through the pairing and gestation period until the F1 generation reached Day 4 post partum (females). At 250 mg AO/kg bw/day one female was found dead at the beginning of the gestation period, but this was not considered to be test item related as no adverse clinical signs were noted prior to death and it was a single case. At 100 and 250 mg AO/kg bw/day all males were found to have reduced activity. Rales and salivation were noted occasionally at 250 mg AO/kg bw/day. In females, rales and salivation were noted in three animals at 250 mg AO/kg bw/day during the gestation period. At 250 mg AO/kg bw/day total locomotor activity was statistically significantly reduced in females. Mean food consumption was dose dependently reduced in males at 100 and 250 mg AO/kg bw/day throughout the study treatment period and in females at the same doses during the pre-pairing and gestation periods. During the lactation period it recovered at 100 mg AO/kg bw/day and remained reduced at 250 mg AO/kg bw/day. Mean body weight and mean body weight gain were reduced throughout the study at 250 mg AO/kg bw/day and during the pre-pairing period at 100 mg AO/kg bw/day for males and at 250 mg AO/kg bw/day throughout the study for females. Effects seen in haematology and clinical biochemistry at 250 mg AO/kg bw/day were not considered to be adverse. An increase in absolute and relative liver weight was noted in males and females at the same dose. At necropsy the mucosa in the forestomach was thickened with irregular surface and a thickened stomach was noted in 5 of 10 males at 250 mg AO/kg bw/day. Lymphoid depletion in the spleen was noted in 3 of 5 females at 250 mg AO/kg bw/day. At the same dose hepatocellular hypertrophy was noted in males (2/5) and females (1/5). In the kidneys of males an increase in the incidence of tubular basophilia and hyaline droplets was recorded in males at all doses. However at 40 and 100 mg AO/kg bw/day the severity of these effects were the same as for control males. In the forestomach hyperkeratosis, parakeratosis, squamous cell hyperplasia and submucosal inflammation were recorded in all males and females at 100 and 250 mg AO/kg bw/day. Submucosal oedema was recorded in males (3/5) and females (3/5) at 250 mg AO/kg bw/day and in females (2/5) at 100 mg AO/kg bw/day. Erosions were recorded in males (3/5) and females (3/5) at 250 mg AO/kg bw/day. Ulcerations were recorded in females at 100 (1/5) and 250 (1/5) mg AO/kg bw/day and pustules in males (2/5) at 250 mg AO/kg bw/day and females (1/5) at 100 and 250 mg AO/kg bw/day. Based on these results, the overall NOAEL was established at 40 mg AO/kg/day.

It can been seen from the results of these two studies that both C12-14 AO and C12-18 AO produce similar effects when dosed by the oral route, primarily driven by local irritative effects in the forestomach. On this basis and given the large overlap in chainlength distributions, similar acute toxicity and physico-chemical properties it is considered that read across to higher tier studies conducted using C12-14 AO is relevant. The available studies for C12 -14 AO are summarised below.

In the key study [Hazelton Laboratories (1974)] using methods comparable to OECD guideline 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents", rats (CD (SD)) received C12 -14 AO via feed at levels of 0, 0.02, 0.1 and 0.5 % for 13 weeks. Twenty animals/sex/group were used. Five males and five females from each treatment group (including untreated control group) were sacrificed at 4 weeks, 10 males and 10 females were sacrificed after 13 weeks, and the remaining animals, after review of the previous necropsy findings, were sacrificed at the end of week 14. The effect of the substance on the rats was evaluated according to physical appearance, behaviour, growth (weekly), food consumption (weekly), survival, clinical laboratory studies (blood and urine), microscopic eye examinations, organ weights, and gross and microscopic pathology. No adverse effects were observed in rats consuming diets containing 0.02 or 0.1% of the substance. Male and female rats receiving diets containing 0.5% test substance generally consumed less diet (grams of diet/kg body weight), and a depression in the average body weight gain was observable by the end of the first week. This sign continued throughout the 14 weeks with growth rates, food consumption, and terminal body weights for these animals being significantly affected. The alterations observed in the absolute and relative organ weights (including the testes and ovaries) in the 0.5% treatment group probably were secondary to body weight effects. Clinical laboratory findings for the high dose group animals generally remained within acceptable limits; however, slight electrolyte imbalance and slight elevation of alkaline phosphatase and blood urea nitrogen values were evident in some animals. Subsequent histopathology failed to confirm an indication of alterations in organ morphology and these findings may be related to the decrease in food consumption. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. Significantly, 2/20 males and 2/20 females in the 0.5% treatment group developed moderate to severe bilateral cataracts. The incidence and early appearance of these cataracts in only the high dose group suggests that these ocular changes may be an effect of test substance treatment. This effect could be a direct effect of the substance or an indirect effect caused by the effect of the test substance on food consumption and nutrient absorption or utilization. Based on these results, the NOAEL for the test substance was 0.1% (in the diet) or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain and age, this translates into a delivered dose of 88 mg AO/kg bw/day.

In a supporting study [Hazelton Laboratories (1980)] using methods comparable to OECD guideline 408 " Repeated Dose 90-day Oral Toxicity Study in Rodents", rats (CD (SD)) received C12 -14 AO via feed at levels of 0, 0.1, 0.2 and 0.4% for 13 weeks. The objective of this study was to establish the dose levels of the test substance that would likely to be maximum tolerated levels throughout a two-year chronic feeding study in rats. Microscopic examination of the tissues for all control males and females, all mid dose females, and from 10 rats/sex from the high dose group did not reveal any compound-related alterations. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. Decreased body weights and lenticular lesions were observed at dose levels of 0.2% in the diet (LOAEL) and above when administered to the rat for 91 days. The NOAEL was determined to be 0.1% in the diet or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain/age, this translates into a delivered dose of 88 mg AO/kg bw/day.

In a supporting study [Batelle (1980)] which was designed to evaluate ocular toxicity of the test substance, Sprague-Dawley CD rats were administered C12 -14 AO via feed at a level of 0.25% for 10 weeks. Rats were ophthalmoscopically examined prior to the study initiation and at study Weeks 6 and 10. Based on the results the LOEL was determined to be 0.25 % based on lenticular opacities in female animals and decreased body weights and body weight gains in both sexes.

In a supporting study [Procter & Gamble (1978)] using methods comparable to OECD guideline 407 "Repeated Dose 28-day Oral Toxicity Study in Rodents" rats (CD (SD)) received the test substance via feed at a level of 0.35% for 6 weeks. The cataractogenic activity of 0.35% commercial test substance in feed was demonstrated. No NOAEL was identified in this study.

In a supporting study [Gibson (1979)] which was designed to evaluate the cataractogenic potential of the test substance, Sprague-Dawley CD rats were administered C12 -14 AO via feed. Dose levels of 0.12%, 0.35% and 0.5% of a commercial grade material and 0.35%, 0.4% and 0.5% of a laboratory prepared material of the test substance was administered in two phases, one for 16 weeks and the other for 30 weeks. No NOAEL/ LOAEL was identified since all doses affected body weight gain, food consumption, and feed efficiency.

In a supporting study [Hazelton Laboratories (1977)] using methods similar to OECD guideline 408 "Repeated Dose 90-day Oral Toxicity Study in Rodents", except using New Zealand white rabbits, C12 -14 AO was administered via feed at levels of 0.1, 0.5 and 1.0 % initially for 13 weeks with the low and mid-dose groups exposed for an additional 19 weeks (total 32 weeks) to evaluate the potential for ocular lesions. Treatment related effects attributable to the administration of the high-dose (1% AO in diet) included increased mortality rates, decreased body weight and food consumption, increased incidences of clinical signs (listlessness, hyperpnea, wheezing and thinness), decreased hemogram (hematocrit, haemoglobin, and erythrocyte) values and increased bilirubin and decreased alkaline phosphatase values. Also, dose-related increases in total bilirubin values were noted at Week 13, and statistically significantly higher liver/body weight ratio were noted in the mid-dose males at Week 32. Gross and microscopic pathology examinations revealed findings attributable to the administration of the high-dose level of the test substance at 1% in diet consisting of gross findings of enlarged cervical and mesenteric lymph nodes, the presence of purulent material (including abscesses) in the lungs, and the absence of body fat and compound-related histomorphologic alterations in the lung, spleen, small intestine and mesenteric lymph node characterized by the presence of large foamy appearing macrophages in these tissues. In addition, marked vacuolation of the bronchial epithelium was present in the lung sections from the high-dosed animals. Based on treatment-related effects on mortality, body weight, hematology, clinical chemistry, and gross and histopathology relative to control at high dose and increased liver/BW ratio at mid dose, the NOAEL for the test substance was 0.1% in diet or 1000 mg AO/kg diet. Using a food consumption factor of 0.032 kg diet/kg bw/day for rabbits, this translates to a dose of 32 mg AO/kg bw/day.

In a sub-acute study [Procter & Gamble (1979)] male rats (Charles River Sprague Dawley derived) were dosed by oral gavage with C12 -14 AO for at 28 days. Each dose was 86 mg of commercial amine oxides (containing approx 27% amine oxide) in 5 ml of water daily by stomach tube which was calculated to be equivalent to the dose received in a previous study which used dietary administration. When the results of the current study were compared with the results of a previous study in which amine oxides were fed in the diet, it was concluded that amine oxides given by stomach tube were not as toxic as the same amount of amine oxide fed in the diet.

Repeated dose toxicity dermal:

No repeated dose dermal toxicity studies are available for C12 -18 AO. Two studies are available for C12 -14 AO. Based on similar acute dermal toxicity (systemic and local) and the similar physico-chemical properties these studies are considered to be relevant for read across to C12 -18 AO. In the key study [Hazelton Laboratories (1978)] using methods comparable to OECD guideline 411 " Subchronic Dermal Toxicity Study: 90-day study", ICR Swiss CD-1 mice received daily (6 hours/day/5 days/week) dermal applications of the test substance at dosage levels of 0.27% (0.27 mg per application) or 1.33% (1.33 mg per application) C12 -14 AO for 91 days. After 28 days (20 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals (15 males and 15 females) continued treatment until the end of the study. No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any substance related lesions with the exception of skin effects at the site of treatment for the 0.27% group after 28 days applications of the test substance. Repeated applications of 1.33% over the 28 days resulted in slight to moderate erythema and scaling in most animals. The histological examinations of the 0.27% dose group after 28 days exhibited dermal irritation consisting of minimal to slight acanthosis. The dermal irritation effects were more pronounced in animals dosed with 1.33% consisting of acanthosis and hyperkeratosis.  As such, a LOEL for localised dermal effects was established of 0.27% test substance for male/female mice.

The second study [Hazelton Laboratories (1976)] was a preliminary sub-acute study conducted to obtain toxicity and mortality data on two sources of the test substance C12 -14 AO for use in setting dose levels in a subsequent chronic study. Initially groups of Charles River ICR Swiss mice received daily (6 hours/day/5 days/week) dermal applications of the test substance at dosage levels of 5, 10, 15 and 20%. However due to severe skin irritation observed at the higher dose levels dosing was terminated. The study was restarted using lower dose levels of 0.5, 1.0 and 2.0% test substance for 4 weeks and a LOEL of 2% and a NOAEL of 1% established. The 2% dose level induced minimal effects in the treatment area of the dorsal skin; however an increase in mortality and some dermal and systemic test substance related effects were seen in some mice at this dose level.

Repeated dose inhalation toxicity:

No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration (90 days) and the highest NOAEL above the lowest LOAEL was chosen (key study).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but, based on the risk assessment, it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but, based on the risk assessment, it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Study with longest duration

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Study was selected as sufficient information was available in the study to determine the amount dosed.

Justification for classification or non-classification

The NOAEL from the key 90 day repeated dose oral toxicity study performed using C12 -14 AO is 88 mg AO/kg bw/day [Hazelton Laboratories (1974)]. Adverse effects were seen at the next level of 440 mg AO/kg bw/day. In a second 90 -day study [Hazelton Laboratories (1980)] an NOAEL of 88 mg AO/kg bw/day was also derived. Adverse effects were seen at the next dose level of 176 mg/kg bw/day. According to the Guidance on the Application of Regulation (EC) No 1272/2008 Table 3.9.3, classification in Category 2 is applicable when significant toxic effects observed in a 90 -day repeated dose study conducted in experimental animals are seen to occur within the guidance value range of 10><=100 mg/kg bw/day. The Guidance also states in Section 3.9.2.3.2 that in the case where the NOAEL is below the guidance value then the effective dose level (ED) should be determined in order to establish the need for classification. In the case where the ED is above the guidance value then interpolation between the ED and the NOAEL is required to determine whether the effects expected at or below the guidance value would warrant classification. In the case of this substance, The NOAEL is 88 mg/kg bw/day and the ED is 176 mg/kg bw/day. There is no evidence to suggest that the dose response curve is steep enough that effects would be seen below 100 mg/kg bw/day, hence classification is not warranted.