N-Phenyl-diethanolamine, reaction products with formaldehyde

Endpoint summary

Description of key information

Acute Toxicity: the test substance could not be classified to rats via the oral and dermal routes, in accordance with the CLP Regulation No 1272/2008. During an acute oral toxicity test (OECD 423) and an acute dermal toxicity test (OECD 402) both reported a LD₅₀ of >2,000 mg/kg bw/day. In both studies, no mortality was observed at dose/concentration of 2000 mg/kg bw/day, respectively. In both tests, hunched posture was noted with uncoordinated movements, decreased activity, shallow breathing observed in acute oral toxicity testing. Local toxicity in the form of slight to well defined erythema was noted between 24 and 72 hours post dosing during acute dermal toxicity testing. Additionally, red discolouration was seen in days 2-7 with flaking, signs of necrosis noted between days 2 and 15.

Local Toxicity: the substance is non-corrosive to the skin in vitro (OECD 431) but irritative to skin (OECD 439) and eye in vitro (OECD 437, 492)_.  The substance caused sensitisation effect in vivo (LLNA – OECD 429) with an EC₃ value reported to be between >0 and 10% (the estimated test item concentration that will give a SI = 3). At 25 and 50% test item concentration no signs of systemic toxicity were noted with very slight irritation observed in the animals between days 1 and 5. No mortality was reported during the main test.  There were no abnormal changes to the body weight of all main test animals.

 

Sub-Acute Toxicity: in a dose range finder for a combined repeated dose toxicity study with the combined repeat dose toxicity study with reproduction/developmental toxicity screening test (OECD 422), where two groups of three female rats were exposed to either a dose level of 500 or 1000 mg/kg/day over a 10 day period. Group one was administered 500 mg/kg bw/day substance (dose concentration: 417 mg/mL), no unscheduled mortality occurred during the study. Salivation was reported for all animals after dosing on days 3, 5 to 10. Macroscopic examinations reported red foci on glandular mucosa of the stomach (2/3 animals)., thickened limited ridge of the stomach (3/3), pale discoloration of the glandular mucosa (1/3 animals).

 

Group two were administered 1000 mg/kg bw/day substance (dose concentration: 833 mg/mL), no unscheduled mortality was noted during the study. Clinical observations of all animals’ report salivation (3/3 animals), hunched posture (3/3 animals), lethargy (2/3 animals), flat posture (1/3 animals), uncoordinated movements (2/3 animals), piloerection (2/3 animals), hypothermia (1/3 animals), deep respiration and ptosis (1/3 animals). Macroscopic examinations reported black and/or red foci on glandular mucosa of the stomach and forestomach, thickening of the stomach wall or limiting ridge and irregular surface of the glandular mucosa and forestomach (3/3 animals) and hardened stomach wall with enlarged liver and spleen (1/3 animals).

 

Slight body weight loss was noted during the study in both group one and two with it being 2% and 3% respectively.

Due to the results observed in group 2 (1,000 mg/kg/day) it was decided that the dose levels for the main OECD 422 study were 50, 150, and 650 mg/kg/day.

 

OECD 422 (combined micronucleus testing; OECD 474): Four groups of 10 male and 10 female Wistar Han rats were administered 0 (control article [dimethylsulfoxide]), 50, 150, or 650 mg/kg/day of the substance once daily for a minimum of 28 days by oral gavage. An additional fifth group of 5 male rats were administered with cyclo-phosphamide (positive control) at a dose of 19 mg/kg/day. Males were dosed for 29 days up to and including the day before scheduled necropsy (this included a minim of 14 days prior to mating and during the mating period)) and were sent to necropsy on Day 30. Females that delivered were treated or 50-64 days (14 days prior to mating, with the objective to cover at least two complete estrous cycles), variable time to conception, the duration of pregnancy and at least 13 days after the delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41 to 44 days. Positive control animals (5 males) were dosed once by oral gavage at 48 (± 1h) hours prior to isolation of bone marrow.

 

Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow were not induced following test article administration at any dose level when compared to vehicle treated animals.

 

No treatment related deaths occurred in the parental study during the study period. However, one pup at 50 mg/kg/day, two pups at 150 mg/kg/day and one pup at 650 mg/kg/day were found dead at first litter check. No toxicological relevance was attributed to these deaths.

Following 650 mg/kg/day, lethargy and flat posture were recorded for 8 out of 10 males and all females, and for each of these animals these signs occurred on multiple days during treatment. A number of these animals also showed hunched posture, rales, piloerection, uncoordinated movements and/or ptosis on a few days during treatment.

 

At 150 mg/kg/day flat posture was noted for one male and two females on treatment Days 12 and/or 13 only. One female at 150 mg/kg/day (No. 65) showed hunched posture, labored respiration, rales and/or piloerection during the last three days of treatment.

One female in dose groups 50 and 150 mg/kg/day showed piloerection on two or three consecutive days during the 6^th treatment week. These incidences were noted as similar to those encountered in the control group where two females showed piloerection (including hunched posture), therefore these were considered not to be related to treatment with the test item.

 

Similarly, to the dose range finder salivation was noted following administration of all dose levels, this was noted as not toxicologically relevant and a common occurrence following oral gavage.

 

In the 650 mg/kg/day dose group the mean body weight gain in male rats was noted to be statistically significantly lower throughout the treatment period with temporary and minimal weight loss of between 2 and 6 % between the first and third treatment week. It was noted that the overall weight gain in these animals were lower than the others within the same group. Additionally, the body weight gain of females was indicated as lower throughout the lactation period with it not noted as being statistically significant) One animal was noted to have no to minimal weight gain during the lactation period with an additional female indicating weight loss over the same period.

 

Slight weight loss was noted in female rats in dose groups. A lower mean body weight gain in males in the 50 and 150 mg/kg/day dose groups was ascribed to slightly higher mean starting body weights (which were not statistically significant at 150 mg/kg/day), with mean body weights during the remainder of the treatment period were similar to those noted in the control group. The mean body weight gain of females in the 150 mg/kg/day dose group was noted to be statistically significantly on post-coitum Days 7, 11 and 14 which continued in the absence of a dose related trend. The variants noted were not ascribed to treatment of the test item with body weights and body weight gain at lower doses of 50 and 150 mg/kg/day considered not to be an affect by treatment with the test item.

 

For animals administered 650 mg/kg/day a number of statistically significant hematological parameters were noted in males and females. For both males and females higher white blood cell count was noted while in males a higher neutrophil count, higher lymphocyte count, lower red blood cell count, higher reticulocyte count, lower hemoglobin and lower mean corpuscular hemoglobin concentration. Clinical biochemistry for males in the same dose group were noted to have statistically significant changes in total bilirubin and urea where both parameters were noted as higher of that of the control.

 

A statistically significant higher liver weight (absolute and relative to body weight) was recorded for males at 650 mg/kg/day. Microscopic evaluation centrilobular hepatocellular hypertrophy. With a statistically significant higher spleen weight (absolute and relative to body weight) also recorded for males in the same group with microscopic evaluation indicating extramedullary hematopoiesis at increased severities compared to control severities.

 

Test item-related microscopic findings following treatment at all dose levels was noted in the stomach and spleen of both males and females and in kidneys and liver of males. Both ulcerative inflammation (to a massive degree) and transmural necrosis (slight or marked degree) was observed in the forestomach of both males and females at 650 mg/kg/day. It was noted that the ulcers were large and accompanied by inflammation mostly in the submucosa and/or at the serosal side with large necrotic areas noted throughout all layers of the forestomach.

 

In the same dose group, the glandular stomach of two males and a single female showed signs of an ulcer or erosion (slight degree) with submucosal inflammation also noted. In a single male at 50 mg/kg/day and three males at 650 mg/kg/day haemorrhages were observed which were linked with the dosing of 650 mg/kg/day where reddish focus/foci were noted in the glandular stomach.

 

A dose-dependent increase in incidence and severity in extramedullary hematopoiesis was observed in males starting at 50 mg/kg/day, up to slight degree, with pigmentation increasing in severity in males and females treated at 650 mg/kg/day. Similarly, in the liver a dose-dependent increased incidence of minimal hepatocellular centrilobular hypertrophy was observed in males starting at 150 mg/kg/day. A combination of tubular findings was observed in kidneys of males treated with 150 and 650 mg/kg/day which consisted of increased incidences and severity of tubular basophilia up to a marked degree, the presence of granular casts and degeneration of tubular epithelial cells to a moderate degree.

 

Reproductive performance was unaffected; the test article did not affect the length and regularity of the estrous cycle, mating index, precoital time, litter size, post-implantation survival index was considered not to have been affected by treatment with the test item.

 

It was concluded that once daily oral gavage administration of 50, 150, or 650 mg/kg/day n-phenyl-diethanolamine, reaction products with formaldehyde to male rats for 29 consecutive days and to female rats for 50-64 and 41-44 days respectively for those who delivered successfully and those that did not deliver. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 50 mg/kg/day based on the adverse degenerative kidney lesions noted in males at 150 mg/kg/day. Reproductive performance and offspring development were unaffected as such, the no observed adverse effect level (NOEL) and NOAEL for reproductive toxicity was considered as 6500 mg/kg/day.

 

Genetic Toxicity: the substance was determined to have genotoxic potential in vitro following a positive result in both OECD 471 (Ames) and OECD 487 (MNT) tests.  The substance was determined not to have a genotoxic potential in vivo following a negative result in OECD 474.

 

Absorption

The molecular weight, vapour pressure, surface tension, water solubility and n-octanol/water partition coefficients of all constituents, 1 to 7, are presented in Table 1. These physicochemical properties are suggestive of favourable absorption via the oral and dermal routes, with the oral uptake more favourable based on the hydrophilic nature of the test substance. Furthermore, being an irritant, gastrointestinal membrane penetration will be enhanced and therefore uptake will be more favourable following oral exposure. The dermal absorption of the test substance is expected due to the high transfer of the substance between the stratum corneum and the epidermis . This is demonstrated by the local toxicity noted in the in vivo sensitisation study on the test substance. For absorption calculation, a default value of 100 % skin absorption according to ECHA 2017 (Chapter R.11). Due to the variety in the number of potential structures related to constituent 1 a conservative approach was taken in the assessment. In this assessment of the absorption capability of the substance it is considered in its entirety and not as individual constituents.

 

Absorption from the gastro-intestinal tract: based on the molecular weight of the majority of the constituents being < 300 g/mol with those having moderate water solubility intracellular and paracellular absorption of the substance is possible based on the results from the in vitro skin irritation study OECD 439. This will result in the substance being delivered into the liver via portal circulation i.e., first pass metabolism. Furthermore, with oral uptake favoured and the test item classified as an irritant this may aid the absorption by causing damage to the membrane resulting in an increased systemic bioavailability of the substance. No unscheduled mortalities were observed in the subacute (OECD 422) study; however, stomach lesion was observed in both male and female rats at the high dose of 650 mg/kg/day. The stomach lesions consisted of crateriform retractions (black and hard) noticeably in the forestomach of 9/10 males and 4/10 females with thickening limiting ridges in 10/10 males and 4/10 females. Ulcerative inflammation of the forestomach was also noted with transmural necrosis, red focus/foci in the glandular stomach was noted in 5/10 males. In the 50 mg/kg/day dose group one single male was noted to have haemorrhages in the glandular stomach. The overt clinical toxicities observed in the sub-acute oral studies in the rat as an indication of the systemic availability of the substance. Based on the vapour pressures of the constituents outlined in Table 1, exposure via inhalation route is possible. Upon inhalation, absorption into pulmonary circulation would follow similar route to oral uptake. However, a high plasma concentration of the substance as well as plasma half-life is expected since the rate fast pass metabolism in the lung is slower than in the liver. For absorption calculation, a default route-to-route extrapolation according to ECHA 2017 (Chapter R.11) can be applied as conservative approach for risk assessment.

 

With a number of constituents in the test substance being of low molecular weight (<100 g/mol) and lacking surface activity it is therefore likely to cause adverse reactions to the skin, as supported by the classification as Skin Irritant 2. Dermal irritation would cause damage that would enhance penetration through the skin, however, transfer between the stratum corneum and the epidermis is low and therefore overall systemic bioavailability of the substance compared to oral exposure.

 

Table 1. Physicochemical properties of constituents 1 – 6*

Constituent	Boiling point	Water Solubility	N-Octanol/water Partition Coefficient	Vapour Pressure	Surface tension
1	No information available	No information available	No information available	No information available	No information available
2	346.73 ºC at 101 325 Pa	48.3 g/L @ 20 ºC	0.63 at 25 ºC	6.2 x10^-6 hPa at 20 ºC	Substance is not surface active
3	≥ - 21 ºC and ≤ -19 ºC at 1013.25 hPa (pure formaldehyde)	550 g/L at 20ºC	0.35 at 25 ºC	Ca 5176 -5186 hPa at 25 ºC (gas)	Substance is not surface active
4	82.5 ± 8.0 ºC (predicted)	No information available	No information available	No information available	No information available
5	64.7 ºC at 101 325 Pa	Miscible	-0.77 at 20 ºC	169.27 hPa at 25 ºC	Substance is not surface active
6	184.4 ºC at 1013 hPa	35 g/L at 20 ºC	0.91 at 25 ºC (pH 7.5)	0.4 hPa at 20 ºC	70.5 mN/m at 20 ºC

^*Constituent 7 was not included in this table as it is an unknown.

 

Distribution

The test substance has physicochemical properties (mainly molecular weight and its water solubility) means upon oral and dermal exposure, the substance can pass through aqueous pores or be carried through the epithelial barrier, via paracellular pathway, into the liver. This will limit distribution of the constituents systemically especially via oral route and therefore resulting into a reduced half-life of the parent compound in blood plasma. However, based on the irritative classification (Skin Irritant 2) of the test substance following oral administration where multiple lesions were noted in both the glandular and forestomach increases the potential of systemic bioavailability of the substance.

 

With the test compound having a moderate water solubility coupled with a low n-octanol/water partition coefficient (as outlined in Table 1) this could result in a high plasma half-life. This means the delivery of the components would be more targeted towards the liver and kidneys which is seen following sub-acute oral exposure in rats. This is supported by the clinical observations of increased liver weight was seen in males in the 650 mg/kg/day group. Systemic distribution via dermal absorption is limited as demonstrated by the lack of systemic toxicity observed during the LLNA. Based on the n-octanol/water partition coefficient, bioaccumulation of the test substance is not expected

 

Metabolism

Metabolism of the test compound is mainly through phase I and II enzymes as demonstrated by increased liver weight and total bilirubin and bile acid in males following sub-acute exposure to the test substance. Metabolism is expected to be rapid with constituent such as constituent 5 (Methanol) being oxidized, mediated by alcohol dehydrogenases to form formaldehyde which in turn is converted to formate and further metabolised to carbon dioxide and water. Test substance metabolites will then undergo further phase II metabolic pathways forming mainly conjugative derivatives of glutathione and eliminated via urine.

 

Excretion

Based on the absorption, distribution and potential metabolic pathways highlighted coupled with available systemic data, the substance will most likely be excreted via bile, faces and urine. This is demonstrated from increased liver and kidney weights and increased total bilirubin, bile acids and neutrophil count noted in the sub-acute study. All constituents are of low – mid molecular weights absorbed via paracellular and intracellular pathways would most likely be excreted via urine as supported by the changes observed in the kidney. Furthermore, the irritating tendency means availability of the substance into the circulatory system is rapid making it readily available for biotransformation into conjugated metabolites which are easily eliminated via bile and urine. Unabsorbed constituent will most likely be eliminated via faeces.  

 

Conclusion

The substance has physico-chemical properties which lead to favourable absorption, distribution, metabolism and elimination (ADME). The physicochemical properties of the constituents will not lead to potential bioaccumulation. Exposure from oral and dermal are most preferred routes and with limited bioavailability via inhalation exposure. This is supported by the adverse effects observed in the acute oral toxicity test OECD 422 and in vitro skin irritation test OECD 439 with no adverse systemic effects observed in the LLNA test OECD 429 study.

 

Site-specific microscopic evidence of irritation at primary exposure site such as GI tract and the skin were observed which demonstrates that there is an irritative potential of the substance at this site when dosed via the aforementioned routes and therefore irritation at the GI tract will affect absorption which was previously expected. This means that paracellular absorption and absorption from the gut into portal vein is favoured and therefore fast pass metabolism, ensuring rapid biotransformation and elimination  via bile and urine.

 

Specific substance toxicity noted in the stomach, liver and kidney, in the form of crater formation reactions with thickened limiting ridge in the forestomach and reddish focus/foci in the glandular stomach, increase organ weight of both the liver and the kidney reported in OECD 422 study are considered to be  treatment related but non-adverse.

 

At a high dose of 650, the substance is not well tolerated as demonstrated by and overt toxicities noted in OECD 422. In the OECD 422 study there were no site-specific accumulation of the substance was noted, no premature death, however clinical signs related to the treatment and effects of toxicological importance to humans were noted. There were also no effects seen on sensory reactivity, grip strength, with higher motor activity noted in males dosed at 650 mg/kg/day. However, mean total movements were considered not to be affected by treatment of the test item. Bodyweight was noted as being affected in the 650 mg/kg/day dose group with the body weight gain in males being significantly lower throughout the treatment period. Food consumption was noted as unaffected in both dose groups. This demonstrates that the toxicokinetics of the substance does pose significant toxicological concern.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100